Cationic emulsion complex adjuvants comprising cyclic dinucleotides, methods of making and use thereof

Abstract

This invention relates to a cationic emulsion complex adjuvant comprising cyclic dinucleotides, metabolizable oils, cationic lipids, and surfactants. The raw materials for the complex adjuvant components of this invention are readily available, and the preparation process is simple and low-cost. The complex adjuvant formed by the cationic emulsion containing cationic lipids and the cyclic dinucleotides of this invention is stable, biocompatible, has few side effects, and can improve immune efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, and specifically relates to a cationic emulsion complex adjuvant containing cyclic dinucleotides, its preparation method, and its application in the fields of immunotherapy and prevention. Background Technology

[0002] Adjuvants are non-specific immunomodulators. Adding adjuvants to vaccines can enhance the strength of the immune response, alter the type of immune response, and prolong the duration of the immune response. Especially for vaccines with weak immunogenicity (such as inactivated vaccines, subunit vaccines, recombinant protein vaccines, and peptide vaccines), the rational use of adjuvants can improve the seroconversion rate of vaccines administered to populations with different immunization backgrounds.

[0003] Emulsion adjuvants are an important branch of novel adjuvants. Emulsions can be used in combination with various weak antigens (recombinant proteins, peptides, etc.) and induce high titers of antigen-specific antibodies. Emulsion adjuvants typically contain an oil phase, an aqueous phase, and an emulsifier. Among these, oil-in-water emulsions are predominantly aqueous, exhibiting high human tolerability and good compatibility with most vaccine antigens.

[0004] Studies have shown that using a single adjuvant alone can induce a weaker immune response that is biased towards a particular type, and has limited effect on improving the strength of antigen response, duration of immunity, and immune tolerance. Vaccines using combined adjuvants, on the other hand, may be able to activate the immune response more comprehensively through synergistic effects.

[0005] The development of compound immune adjuvants by combining different types of adjuvants has become a new trend in adjuvant development. However, the question remains: whether the combined application of adjuvants with different mechanisms is complementary, what changes will occur in their safety and adjuvant activity, what proportions of each adjuvant component should be used, and what formulation process can maximize immunostimulatory activity while ensuring safety. These questions are unpredictable and require experimental verification. They have become technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a biocompatible compound adjuvant that can enhance the immune effect of vaccines.

[0007] To achieve the above objectives, the present invention provides a cationic emulsion complex adjuvant comprising cyclic dinucleotides, metabolizable oils, cationic lipids, and surfactants.

[0008] In some embodiments, the cyclic dinucleotide is selected from at least one of 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP, c-di-AMP, ADU-S100, and their salts.

[0009] In some embodiments, the cationic lipid is selected from one or more of DOTMA, DOTAP, DODMA, SM-102, ALC-0315, ALC-0519, C12-200, DlinDMA, and DLin-MC3-DMA.

[0010] In some embodiments, the metabolizable oil is squalene.

[0011] In some embodiments, the surfactant comprises one or more of the following: polyoxyethylene sorbitol fatty acid ester (Tween), sorbitol fatty acid ester (Span), Triton X-100 (polyethylene glycol octylphenyl ether), and lecithin.

[0012] In some embodiments, the surfactant comprises one or both of Tween 80 and Span 85.

[0013] In some embodiments, the compound adjuvant further comprises α-tocopherol.

[0014] In some embodiments, the compound adjuvant comprises a cyclic dinucleotide, a cationic lipid, squalene, α-tocopherol, and Tween 80.

[0015] In some embodiments, the compound adjuvant comprises 1-500 μg / mL of cyclic dinucleotide, 0.01-2 mg / mL of cationic lipid, 2-50 mg / mL of squalene, 2-50 mg / mL of α-tocopherol and 1-50 mg / mL of Tween 80.

[0016] In some embodiments, the content of cationic lipids in the compound adjuvant is selected from 0.01-2 mg / mL, for example 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, or 2 mg / mL.

[0017] In some embodiments, the squalene content in the compound adjuvant is selected from 2-50 mg / mL, for example 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, etc. mg / mL, 41 mg / mL, 42 mg / mL, 42.75 mg / ml, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, or 50 mg / mL.

[0018] In some embodiments, the α-tocopherol content in the compound adjuvant is selected from 2-50 mg / mL, for example, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, etc. mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 47.5 mg / ml, 48 mg / mL, 49 mg / mL, or 50 mg / mL.

[0019] In some embodiments, the content of Tween 80 in the compound adjuvant is selected from 1-50 mg / mL, for example, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 3 ... mg / mL, approximately 34 mg / mL, approximately 35 mg / mL, approximately 36 mg / mL, approximately 37 mg / mL, approximately 38 mg / mL, approximately 39 mg / mL, approximately 40 mg / mL, approximately 41 mg / mL, approximately 42 mg / mL, approximately 43 mg / mL, approximately 44 mg / mL, approximately 45 mg / mL, approximately 46 mg / mL, approximately 47 mg / mL, approximately 48 mg / mL, approximately 49 mg / mL, or approximately 50 mg / mL.

[0020] In some embodiments, the content of the cyclic dinucleotide in the compound adjuvant is selected from 1-500 μg / mL, for example 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 187.5 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL. , 230μg / mL, 240μg / mL, 250μg / mL, 260μg / mL, 270μg / mL, 280μg / mL, 290μg / mL, 300μg / mL, 310μg / mL, 320μg / mL, 330μg / mL, 340μg / mL, 350μg / mL, 360μg / mL , 370μg / mL, 380μg / mL, 390μg / mL, 400μg / mL, 410μg / mL, 420μg / mL, 430μg / mL, 440μg / mL, 450μg / mL, 460μg / mL, 470μg / mL, 480μg / mL, 490μg / mL or 500μg / mL.

[0021] In some embodiments, the human dose of the compound adjuvant is 0.1-1 mL, preferably 0.4-1 mL.

[0022] In some embodiments, the human dose of the compound adjuvant comprises: 2-20 mg squalene, 2-20 mg α-tocopherol, 1-20 mg Tween 80, 0.01-1 mg cationic lipid, and 1-500 μg cyclic dinucleotide. Preferably, the human dose of the compound adjuvant comprises 5-15 mg squalene, 5-15 mg α-tocopherol, 2-15 mg Tween 80, 0.1-0.5 mg cationic lipid, and 20-200 μg cyclic dinucleotide.

[0023] In some embodiments, the human dose of the compound adjuvant comprises: 2-20 mg squalene, 2-20 mg α-tocopherol, 1-20 mg Tween 80, 0.01-1 mg DOTAP, and 1-500 μg c-di-GMP. In some embodiments, the human dose of the compound adjuvant comprises 2-20 mg squalene, for example, 2 mg, 3 mg, 4 mg, 5 mg, 5.13 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 10.26 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In some embodiments, the human dose of the compound adjuvant contains 2-20 mg of α-tocopherol, such as 2 mg, 3 mg, 4 mg, 5 mg, 5.7 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 11.4 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In some embodiments, the human dose of the compound adjuvant contains 1-20 mg of Tween 80, such as about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg. In some embodiments, the human dose of the compound adjuvant contains 0.01-1 mg of DOTAP, for example, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg, 0.12 mg, 0.2 mg, 0.24 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg.In some embodiments, the human dose of the compound adjuvant contains 1-500 μg c-di-GMP, for example 1 μg, 5 μg, 9 μg, 10 μg, 20 μg, 22.5 μg, 30 μg, 40 μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 187.5 μg, 190 μg, 200 μg, 210 μg, 220 μg, 2... 30μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg, 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg or 500μg.

[0024] In some embodiments, the human dose of the compound adjuvant contains 5-15 mg squalene, 5-15 mg α-tocopherol, 2-15 mg Tween 80, 0.1-0.5 mg DOTAP, and 20-200 μg c-di-GMP.

[0025] In some embodiments, the complex adjuvant further comprises monophospholipid A.

[0026] In some embodiments, the monophosphoryl lipid A is 3D-MPL.

[0027] In some embodiments, the content of 3D-MPL is 1-500 μg / mL, for example 1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, and 450 μg / mL.

[0028] In some embodiments, the human dose of the compound adjuvant contains 1-500 μg of 3D-MPL, preferably 20-200 μg, and most preferably 20-100 μg.

[0029] In some embodiments, the aqueous phase is a phosphate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, an acetate buffer solution, or a citrate-phosphate buffer solution.

[0030] On the other hand, the present invention provides a method for preparing the composite adjuvant, comprising mixing an oil phase containing cationic lipids and metabolizable oils with an aqueous phase containing surfactants, emulsifying and homogenizing the mixture to form a cationic emulsion, and mixing the cationic emulsion with a cyclic dinucleotide to obtain the composite adjuvant.

[0031] In some embodiments, the aqueous phase is a phosphate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, an acetate buffer solution, or a citrate-phosphate buffer solution.

[0032] In some embodiments, the oil phase further comprises α-tocopherol.

[0033] In some embodiments, the oil phase further comprises monophosphoryl lipid A.

[0034] In some embodiments, the composite adjuvant is obtained by mixing a cationic emulsion with a cyclic dinucleotide, comprising particles dispersed in an aqueous phase and a cyclic dinucleotide, said particles comprising a cyclic dinucleotide, a metabolizable oil, a cationic lipid, and a surfactant. In some embodiments, the particles further comprise α-tocopherol. In some embodiments, the particles further comprise monophospholipid A.

[0035] The present invention also provides another method for preparing the composite adjuvant, comprising mixing an oil phase containing cationic lipids and metabolizable oils with an aqueous phase containing surfactants and cyclic dinucleotides, emulsifying the mixture, and then homogenizing it to obtain the composite adjuvant.

[0036] In some embodiments, the emulsification speed is selected from 5000-15000 rpm, the dispersion time is selected from 10-30 min, preferably the emulsification speed is 10000 rpm, and preferably the dispersion time is 20 min. In some embodiments, the homogenization pressure is selected from 50-150 MPa, preferably 100 MPa.

[0037] In some embodiments, the aqueous phase is a phosphate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, an acetate buffer solution, or a citrate-phosphate buffer solution.

[0038] In some embodiments, the oil phase further comprises α-tocopherol.

[0039] In some embodiments, the oil phase further comprises monophosphoryl lipid A.

[0040] In some embodiments, a composite adjuvant is obtained by emulsifying and homogenizing an oil phase comprising cationic lipids and metabolizable oils with an aqueous phase containing surfactants and cyclic dinucleotides, and then comprising particles dispersed in the aqueous phase, the particles comprising squalene, DOTAP, c-di-GMP, and Tween 80.

[0041] In some embodiments, the particles further comprise α-tocopherol. In some embodiments, the particles further comprise monophospholipid A.

[0042] In some embodiments, a composite adjuvant obtained by emulsifying and homogenizing an oil phase containing cationic lipids and metabolizable oils with an aqueous phase containing surfactants and cyclic dinucleotides can significantly improve the stability and sustained-release properties of the adjuvant compared with a composite adjuvant obtained by mixing cationic emulsions and cyclic dinucleotides, and the distribution of cyclic dinucleotides in the adjuvant is more uniform.

[0043] In another aspect, the present invention also provides the use of the aforementioned compound adjuvant in the preparation of medicaments for the prevention or treatment of diseases.

[0044] In another aspect, the present invention also provides a vaccine composition comprising one or more antigens and the aforementioned adjuvant.

[0045] In some embodiments, the antigen is one or more antigens derived from viruses, bacteria, fungi, parasites, or tumors.

[0046] In some embodiments, the antigen is derived from at least one of human papillomavirus (HPV), enterovirus that causes hand-foot-mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, novel coronavirus (SARS-CoV-2), hepatitis virus, rabies virus, and metapneumovirus.

[0047] Compared with the prior art, the present invention has the following beneficial effects: The composite adjuvant of the present invention is based on an oil-in-water emulsion, which has good biocompatibility, and the raw materials of each component are relatively easy to obtain, and the preparation process is simple and low in cost. This invention employs a composite adjuvant formed from cationic emulsion and cyclic dinucleotide, which is stable and has few side effects. The cyclic dinucleotide and cationic emulsion work synergistically to enhance the immune effect. Attached Figure Description

[0048] The following figures are intended only to illustrate and explain the present invention and do not limit the scope of the invention. Wherein: Figure 1 The results of cytokine detection in Example 6 of this invention; Figure 2 This is the result of antigen-specific IgG antibody detection in Example 6 of the present invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0050] In this specification, the reference to "one embodiment" means that the specific parameters, steps, etc., described in that embodiment are included in at least one embodiment according to the present invention. Therefore, the use of terms such as "according to one embodiment of the present invention" or "in one embodiment" in this specification does not specifically refer to the same embodiment, and the use of terms such as "in another embodiment," "different embodiments of the present invention," or "other embodiments of the present invention" does not specifically mean that the mentioned features can only be included in specific different embodiments. Those skilled in the art should understand that the specific parameters, steps, etc., disclosed in one or more embodiments of this specification can be combined in any suitable manner.

[0051] This invention provides a compound adjuvant comprising cyclic dinucleotides, metabolizable oils, cationic lipids, and surfactants.

[0052] STING is a receptor-recognized nucleic acid distinct from TLRs (Toll-like receptors). Examples of recognized natural ligands include bacterial / protozoan-derived cyclic dinucleotides (CDNs) such as cyclic guanosine diphosphate (c-di-GMP), cyclic adenosine diphosphate (c-di-AMP), and cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which are metabolites produced by many bacterial pathogens. STING is activated by these natural ligands, inducing phosphorylation of downstream TBK1 (TANK-binding kinase 1) and activating further downstream IRF3 (interferon regulator 3) and NF-κB signaling, thereby inducing a type I interferon (IFN) response. Despite its promising prospects, direct in vivo administration of STING agonists often faces challenges, including poor pharmacokinetic characteristics of CDNs and the toxicity of other small-molecule STING agonists upon systemic exposure. These factors underscore the urgent need for adjuvant systems-delivery platforms that improve stability and in vivo half-life and enable potential targeted delivery.

[0053] The cyclic dinucleotide in this invention is a cyclic molecule composed of two nucleoside monophosphates linked by two phosphodiester bonds, and can be selected from at least one of 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP, and c-di-AMP. The structural formula of cyclic adenosine dimonophosphate is as follows:

[0054] The structural formula of cyclic guanosine diphosphate is:

[0055] The structural formula of cyclic guanosine monophosphate-adenosine monophosphate is:

[0056] In the embodiments of the present invention, the cyclic diadenosine monophosphate used is a disodium salt form with the same biological activity and greater stability, and both were purchased from MedChemExpress.

[0057] In some embodiments, the compound adjuvant contains 1-500 μg / mL of cyclic dinucleotide. The human dose of the compound adjuvant contains 1-500 μg of cyclic dinucleotide. Preferably, the human dose of the compound adjuvant contains 20-200 μg of cyclic dinucleotide.

[0058] The particles of this invention are generally provided in the form of an oil phase, comprising a cyclic dinucleotide, a metabolizable oil, a cationic lipid, and a surfactant, dispersed in an aqueous phase to form a cationic emulsion. In some embodiments, the metabolizable oil is preferably squalene, the cationic lipid is preferably DOTAP, and the surfactant is preferably Tween 80. In some embodiments, the particles also contain α-tocopherol. In some embodiments, the particles also contain MPL.

[0059] The cationic lipids in this invention may be selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-dimethylamino-propane (DODMA), SM-102, ALC-0315, ALC-0519, C12-200, DlinDMA, and DLin-MC3-DMA.

[0060] In some embodiments, the surfactant comprises one or more of polyoxyethylene sorbitan fatty acid ester (Tween), sorbitan fatty acid ester (Span), octylphenyl polyol-9 (Trioton X-100 or polyethylene glycol octylphenyl ether), and lecithin. In some embodiments, the surfactant comprises one or both of Tween 80 and Span 85.

[0061] In a preferred embodiment, the compound adjuvant further comprises α-tocopherol.

[0062] In some embodiments, the aqueous phase is a phosphate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, an acetate buffer solution, or a citrate-phosphate buffer solution.

[0063] In some embodiments, the compound adjuvant comprises a cyclic dinucleotide, a cationic lipid, squalene, α-tocopherol, and Tween 80. The compound adjuvant may contain 1-500 μg / mL of cyclic dinucleotide, 0.01-2 mg / mL of cationic lipid, 2-50 mg / mL of squalene, 2-50 mg / mL of α-tocopherol, and 1-25 mg / mL of Tween 80. In some embodiments, the cationic emulsion can be used as an adjuvant for human vaccines, with a human dose of 0.1-1 mL, wherein each human dose contains 1-500 μg of cyclic dinucleotide, 0.01-1 mg of cationic lipid, 2-20 mg of squalene, 2-20 mg of α-tocopherol, and 1-10 mg of Tween 80. Preferably, the human dose of the compound adjuvant contains 1-200 μg of cyclic dinucleotide, 0.1-0.5 mg of cationic lipid, 5-15 mg of squalene, 5-15 mg of α-tocopherol and 2-10 mg of Tween 80.

[0064] In some embodiments, the complex adjuvant comprises a cyclic dinucleotide, a cationic lipid, squalene, Span 85, and Tween 80. In some embodiments, the complex adjuvant may comprise 1-500 μg / mL of a cyclic dinucleotide, 0.01-2 mg / mL of a cationic lipid, 2-50 mg / mL of squalene, 1-25 mg / mL of Span 85, and 1-25 mg / mL of Tween 80.

[0065] In some embodiments, the cationic emulsion can be used as an adjuvant for human vaccines, with a human dose of 0.1-1 mL, wherein each human dose contains 1-500 μg of cyclic dinucleotide, 0.1-1 mg of cationic lipid, 2-20 mg of squalene, 1-10 mg of Span 85, and 1-10 mg of Tween 80. Preferably, the human dose of the compound adjuvant contains 1-200 μg of cyclic dinucleotide, 0.1-0.5 mg of cationic lipid, 5-15 mg of squalene, 2-10 mg of Span 85, and 2-10 mg of Tween 80.

[0066] In some embodiments, the complex adjuvant further comprises monophospholipid A.

[0067] The monophosphoryl lipid A (MPL) in this application is a non-toxic derivative of lipopolysaccharide (LPS) from Gram-negative bacteria such as Salmonella minnesota R595. It retains the adjuvant properties of LPS while exhibiting reduced toxicity (Johnson et al. 1987 Rev. Infect. Dis. 9 Suppl: S512-S516). 3D-MPL is 3-O-deacylated monophosphoryl lipid A (or 3-de-O-acylated monophosphoryl lipid A). Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A having 4, 5, or 6 acylated chains. In embodiments of the invention, the adjuvant comprises 3-O-deacylated monophosphoryl lipid A (3D-MPL). In some embodiments, the content of 3D-MPL is 10-500 μg / mL, preferably 20-200 μg / mL.

[0068] The compound adjuvant described in this invention can be combined with at least one antigen to form a vaccine composition.

[0069] In some embodiments, the antigen is derived from at least one of human papillomavirus (HPV), enterovirus that causes hand-foot-mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, novel coronavirus (SARS-CoV-2), hepatitis virus, rabies virus, and metapneumovirus.

[0070] The antigens derived from human papillomavirus (HPV) are the L1 and / or L2 proteins of various HPV types. In embodiments of the present invention, the HPV can be low-risk HPV (e.g., HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89), intermediate-risk HPV (e.g., HPV26, 53, 66, 73, 82), or high-risk HPV (e.g., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68).

[0071] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from one or more L1 and / or L2 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58.

[0072] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from the L1 and / or L2 proteins of HPV types 6 and 11.

[0073] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from the L1 and / or L2 proteins of HPV types 16 and 18.

[0074] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from the L1 and / or L2 proteins of HPV types 6, 11, 16, and 18.

[0075] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from the L1 and / or L2 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58.

[0076] In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from the L1 and / or L2 proteins of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.

[0077] Enteroviruses that cause hand-foot-mouth disease mainly include Coxsackievirus A4, 5, 6, 7, 9, 10, and 16, Coxsackievirus B2, 5, and 13, and Enterovirus 71 (EV71). In embodiments of the present invention, the antigens derived from these enteroviruses can be from one or any combination of the above types, and the antigens are preferably virus-like particles (VLPs) composed of VP1, VP2, VP3, and VP4 proteins. VP1, VP2, VP3, and VP4 proteins are produced by the degradation of precursor protein P1 under the action of 3CD protease.

[0078] In some embodiments, the antigens derived from these enteroviruses comprise one or more of EV71, Coxsackie A6, 10, and 16 viral particles. In a preferred embodiment, the antigens derived from these enteroviruses comprise EV71, Coxsackie A6, 10, and 16 viral particles.

[0079] The protein families with strong immunogenicity in Mycobacterium tuberculosis mainly include the Esx family proteins, the PE / PPE family proteins, and the DosR family proteins. Preferred Esx family proteins include ESAT-6, CFP-10, TB9.8, TB10.3, TB10.4, TB11.0, and TB12.9. Preferred PE / PPE family proteins include PPE17, PPE18, PPE34, PPE42, PPE57, PE-PGRS33, PE35-PPE68, PE-PGRS62, PE-PGRS17, PE-PGRS11, and PE25-PPE41. Preferred DosR family proteins include Rv2626c, Rv2029c, Rv2031c, Rv2627c, and Rv3133c.

[0080] In a preferred embodiment of the present invention, the antigen derived from Mycobacterium tuberculosis comprises at least one Esx family protein, at least one PE / PPE family protein, and at least one DosR family protein. In some embodiments, the Esx family protein is CFP-10, the PE / PPE family protein is PE35 and PPE68, and the DosR family protein is selected from Rv2626c, Rv2627c, and Rv2031c. In a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises CFP-10 protein, PE35 protein, PPE68 protein, and Rv2627c protein. In a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein, and Rv2627c protein. In a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises CFP-10 protein, PE35 protein, PPE68 protein, and Rv2626c protein. In one preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein, and Rv2626c protein. In another preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises CFP-10 protein, PE35 protein, PPE68 protein, and Rv2031c protein. In yet another preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein, and Rv2031c protein.

[0081] Antigens derived from herpes simplex virus (HSV) can be gB, gC, gD, gH, gL, gI, ICP0, ICP4, etc., from HSV-1 and / or HSV-2. In embodiments of the present invention, the antigen derived from herpes simplex virus (HSV) comprises the HSV gB protein or a functional fragment thereof. In a preferred embodiment, the antigen derived from herpes simplex virus (HSV) comprises the extracellular domain of the gB protein or a functional fragment thereof. In one embodiment, the functional fragment of the extracellular domain of the gB protein comprises the fusion-loop domain of the gB protein. In one embodiment, the extracellular domain of the gB protein comprises at least one amino acid mutation, preferably, the amino acid mutation is a proline substitution. In one embodiment, the extracellular domain of the gB protein of HSV-1 comprises a proline substitution at position 406. In one embodiment, the extracellular domain of the gB protein of HSV-2 comprises a proline substitution at position 408. In one embodiment, the antigen derived from herpes simplex virus (HSV) comprises a fusion protein formed by the fusion loop domain of HSV-1 gB protein and the fusion loop domain of HSV-2 gB protein.

[0082] Antigens derived from varicella-zoster virus (VZV) include the gB, gC, gE, gH, gI, gK, and gL proteins of VZV. In a preferred embodiment, the antigen derived from varicella-zoster virus (VZV) includes a truncated gE protein that lacks the carboxyl-terminal hydrophobic anchor region of the gE protein.

[0083] Antigens derived from influenza viruses include inactivated influenza virus, hemagglutinin (HA protein) of influenza virus, or neuraminidase (NA protein) of influenza virus. In a preferred embodiment, the antigen derived from influenza virus includes hemagglutinin (HA protein) of influenza virus. In another preferred embodiment, the antigen derived from influenza virus includes inactivated influenza virus.

[0084] Antigens derived from the novel coronavirus (SARS-CoV-2) comprise the SARS-CoV-2 spike protein (S protein), its receptor-binding domain (RBD), or a functionally active fragment thereof. In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) is a fusion protein formed by the receptor-binding domain (RBD) or a functionally active fragment thereof of the SARS-CoV-2 spike protein (S protein) and its N-terminal domain (NTD) or a functionally active fragment thereof. In a preferred embodiment, the fusion protein further comprises a foldon domain, the Fc domain of human immunoglobulin, or a functionally active fragment thereof. In a more preferred embodiment, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises fusion proteins from different strains, each fusion protein being a fusion protein formed by the receptor-binding domain (RBD) or a functionally active fragment thereof, the N-terminal domain (NTD), and the foldon domain or a functionally active fragment thereof. In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises a fusion protein derived from an immune-dominant strain and a fusion protein derived from an epidemic-dominant strain, wherein the immune-dominant strain comprises at least one of a prototype strain and a Beta strain, and the epidemic-dominant strain comprises at least one of a Delta strain and an Omicron strain.

[0085] In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises a fusion protein formed by a receptor-binding domain or a functionally active fragment of the S protein derived from an immunodominant strain and a receptor-binding domain or a functionally active fragment of the S protein derived from a prevalent dominant strain. The immunodominant strain comprises at least one of a prototype strain and a Beta strain, and the prevalent dominant strain comprises at least one of a Delta strain and an Omicron strain. The Omicron strain includes BA.1, BA.2, BA.3, BA.4, and BA.5 variants.

[0086] Human hepatitis viruses include hepatitis A, B, C, D, E, and G viruses. In some embodiments, the antigens derived from the hepatitis viruses include hepatitis B surface antigen (HBsAg) derived from hepatitis B.

[0087] Antigens derived from rabies virus include inactivated rabies virus or recombinant proteins derived from rabies virus. Recombinant proteins are derived from at least one of the rabies virus G protein, N protein, M protein, P protein, and L protein.

[0088] Antigens derived from human metapneumovirus (hMPV) are typically the F protein or fragments thereof, preferably the pre-fusion conformation of the F protein or fragments thereof. In some embodiments, the antigen derived from human metapneumovirus (hMPV) is a virus-like particle formed from the F protein. In some embodiments, the antigen derived from human metapneumovirus (hMPV) further includes a foldon domain.

[0089] Example 1 Preparation of compound adjuvant Weigh 3.42 g of squalene and 3.80 g of α-tocopherol, mix well to form the oil phase. Weigh 80 mg of DOTAP and dissolve it in the oil phase. Take 1.5 mL of 10 mg / mL c-di-GMP solution, add 73.5 mL of phosphate buffer containing 5 wt.% Tween 80, and mix to form the aqueous phase. Add the aqueous phase to the oil phase, disperse at 10,000 rpm for 20 min, and homogenize at 100 MPa to obtain the composite emulsion stock solution. The composite emulsion stock solution contains 42.75 mg / mL squalene, 47.5 mg / mL α-tocopherol, 1 mg / mL DOTAP, and 187.5 μg / mL c-di-GMP. Before use, dilute with water for injection 1-10 times. Example 2 Preparation of compound adjuvant

[0090] Weigh 3.42 g of squalene and 3.80 g of α-tocopherol, and mix well to form the oil phase. Weigh 80 mg of DOTAP and 16 mg of 3D-MPL, and dissolve them in the oil phase. Take 1.5 mL of 10 mg / mL c-di-GMP solution, add 73.5 mL of phosphate buffer containing 5 wt.% Tween 80, and mix to form the aqueous phase. Add the aqueous phase to the oil phase, disperse at 8000 rpm for 20 min, and homogenize at 100 MPa to obtain the composite emulsion stock solution. The composite emulsion stock solution contains 42.75 mg / mL squalene, 47.5 mg / mL α-tocopherol, 1 mg / mL DOTAP, 187.5 μg / mL c-di-GMP, and 200 μg / mL 3D-MPL. Before use, it can be diluted 1-10 times with water for injection. Example 3 Preparation of compound adjuvant

[0091] Weigh 3.42 g of squalene and 3.80 g of α-tocopherol, mix well to obtain the oil phase. Weigh 80 mg of DOTAP and dissolve it in the oil phase. Take 75 mL of phosphate buffer containing 5 wt.% Tween 80 as the aqueous phase, add the aqueous phase to the oil phase, disperse at 10000 rpm for 20 min, and homogenize at 100 MPa to obtain a cationic emulsion.

[0092] Then add 1.5 mL of 10 mg / mL c-di-GMP solution to obtain the composite emulsion stock solution. The composite emulsion stock solution contains 42.75 mg / mL squalene, 47.5 mg / mL α-tocopherol, 1 mg / mL DOTAP, and 187.5 μg / mL c-di-GMP. Before use, it can be diluted 1-10 times with water for injection.

[0093] Comparative Example 1: Preparation of Cationic Emulsions Weigh 3.42 g of squalene and 3.80 g of α-tocopherol, mix well to obtain the oil phase. Weigh 80 mg of DOTAP and dissolve it in the oil phase. Take 75 mL of aqueous buffer containing 5 wt.% Tween 80 as the aqueous phase. Add the aqueous phase to the oil phase, disperse at 10,000 rpm for 20 min, and homogenize at 100 MPa to obtain the cationic emulsion stock solution. The cationic emulsion stock solution contains 42.75 mg / mL squalene, 47.5 mg / mL α-tocopherol, and 1 mg / mL DOTAP. Before use, it can be diluted 1-10 times with water for injection. Comparative Example 2: Preparation of a cationic lipid complex emulsion

[0094] Weigh 3.42 g of squalene and 3.80 g of α-tocopherol, mix well to obtain the oil phase. Take 75 mL of aqueous buffer containing 5 wt.% Tween 80 as the aqueous phase. Add the aqueous phase to the oil phase, disperse at 10000 rpm for 20 min, and homogenize at 100 MPa to obtain the emulsion stock solution. Then add 1.5 mL of 10 mg / mL c-di-GMP solution to obtain a cationic lipid-free composite emulsion stock solution. The composite emulsion stock solution contains 42.75 mg / mL squalene, 47.5 mg / mL α-tocopherol, and 187.5 μg / mL c-di-GMP. Before use, dilute with water for injection 1-10 times.

[0095] Example 4: Preparation of VZV vaccine composition Take the stock solution of the compound adjuvant from Example 1 and dilute it with 1-5 times its volume of water for injection. Using the diluted compound adjuvant, reconstitute the varicella-zoster virus gE recombinant protein, and then add 1-5 times its volume of sodium chloride injection to obtain the VZV vaccine composition. Store at 2-8°C, protected from light. The human dose of the VZV vaccine composition contains 20-50 μg of VZV gE protein and 9-100 μg of c-di-GMP.

[0096] Take the stock solution of the compound adjuvant from Example 2 and dilute it with 1-5 times its volume of water for injection. Using the diluted compound adjuvant, reconstitute the varicella-zoster virus gE recombinant protein, and then add 1-5 times its volume of sodium chloride injection to obtain the VZV vaccine composition. Store at 2-8°C, protected from light. The human dose of the VZV vaccine composition contains 20-50 μg of VZV gE protein, 9-100 μg of c-di-GMP, and 10-100 μg of 3D-MPL.

[0097] Take the stock solution of the compound adjuvant from Comparative Example 1 and dilute it with 1-5 times its volume of water for injection. Using the diluted compound adjuvant, reconstitute the varicella-zoster virus gE recombinant protein, and then add 1-5 times its volume of sodium chloride injection to obtain the VZV vaccine composition. Store at 2-8°C, protected from light. The human dose of the VZV vaccine composition contains 20-50 μg of VZV gE protein.

[0098] Take the stock solution of the compound adjuvant from Example 3 and dilute it with 1-5 times its volume of water for injection. Using the diluted compound adjuvant, reconstitute the varicella-zoster virus gE recombinant protein, and then add 1-5 times its volume of sodium chloride injection to obtain the VZV vaccine composition. Store at 2-8°C, protected from light. The human dose of the VZV vaccine composition contains 20-50 μg of VZV gE protein and 9-100 μg of c-di-GMP.

[0099] Stability considerations in Example 5 1 ml of the emulsions from Examples 1, 3, and 2 were placed in 30 kDa ultrafiltration tubes and centrifuged at 3000 rpm for 30 min to obtain aqueous filtrate and emulsion retrieval liquid, respectively. The content of c-di-GMP in the aqueous filtrate was detected by high performance liquid chromatography to determine the binding rate of c-di-GMP to the oil phase under different preparation methods.

[0100] Table 1. Combination rate of c-di-GMP with oil in composite emulsions with different formulations or preparation methods.

[0101] The experimental results are shown in Table 1. Compared with the composite emulsion without cationic lipids (Comparative Example 2), the binding rate of c-di-GMP to the oil phase was significantly increased to over 85% in the emulsion with added cationic lipids, regardless of whether c-di-GMP was added to the aqueous phase first or mixed with the prepared cationic emulsion. This indicates that the addition of cationic lipids helps c-di-GMP to be stably bound in the oil phase. Furthermore, compared with the preparation method of preparing a cationic emulsion and then mixing it with c-di-GMP (Example 3), the preparation method of adding c-di-GMP to the aqueous phase first (Example 1) resulted in a higher binding rate of c-di-GMP to the oil, indicating that the active ingredient c-di-GMP in the emulsion obtained by this method exists within the nanoparticle droplets.

[0102] Therefore, compared to composite emulsions without cationic lipids or composite emulsions post-mixed with c-di-GMP, the preparation of cationic composite emulsions by adding c-di-GMP to the aqueous phase has the following advantages: 1. Uniform distribution: c-di-GMP directly participates in the emulsification process, resulting in a more uniform distribution; 2. Good protection: The emulsion structure provides physical protection for c-di-GMP, reducing contact with the external environment and improving chemical stability; 3. Controllable release: Achieves stable and sustained release of c-di-GMP, prolonging the adjuvant effect.

[0103] Table 2 Stability Considerations

[0104] Example 6 Immunological Evaluation Using 6-8 week old C57BL / 6N mice as an animal model, the immunogenicity of recombinant varicella-zoster virus gE protein was studied using this antigen. The effect of the adjuvant compound on immunogenicity was investigated, with c-di-GMP and saline as controls. The immunization regimen is shown in Table 3 (immunization dose is 1 / 10 of the human dose). Six mice in each group were immunized with the recombinant varicella-zoster vaccine by intramuscular injection of 0.1 mL on days 0 and 14. On day 28, intracellular cytokine detection and antigen-specific IgG antibody detection were performed to evaluate the cellular and humoral immune effects of the vaccine.

[0105] Table 3 Immunization Schedule

[0106] Cytokine test results as follows Figure 1As shown, the cytokine levels in the cationic emulsion containing c-di-GMP were significantly higher than those in the phosphate buffered saline solution containing c-di-GMP. Surprisingly, when the c-di-GMP content was reduced (Group 2), the cytokine levels were actually higher than in Group 1. No significant changes in cytokine levels were observed when c-di-GMP and 3D-MPL were used in combination.

[0107] Antigen-specific IgG antibody test results as follows Figure 2 As shown, when a cationic emulsion complex adjuvant containing 2.25 μg c-di-GMP is used, the antigen-specific IgG antibody titer is significantly higher than that when phosphate buffer containing c-di-GMP is used as an adjuvant. This indicates that the cationic emulsion and c-di-GMP have a strong synergistic enhancement effect.

[0108] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cationic emulsion composite adjuvant, characterized in that, It contains cyclic dinucleotides, metabolizable oils, cationic lipids, and surfactants.

2. The compound adjuvant according to claim 1, characterized in that, The cyclic dinucleotide is selected from at least one of 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP, c-di-AMP, ADU-S100 and their salts.

3. The compound adjuvant according to claim 1, characterized in that, The cationic lipid is selected from one or more of DOTMA, DOTAP, DODMA, SM-102, ALC-0315, ALC-0519, C12-200, DlinDMA, and DLin-MC3-DMA.

4. The compound adjuvant according to claim 1, characterized in that, The metabolizable oil is squalene.

5. The compound adjuvant according to claim 1, characterized in that, The surfactant comprises one or more of the following: polyoxyethylene sorbitol fatty acid ester, sorbitol fatty acid ester, Triton X-100, and lecithin.

6. The compound adjuvant according to claim 5, characterized in that, The surfactant comprises one or both of Tween 80 and Span 85.

7. The compound adjuvant according to claim 1, characterized in that, The compound adjuvant further comprises α-tocopherol.

8. The compound adjuvant according to claim 7, characterized in that, The compound adjuvant contains cyclic dinucleotides, cationic lipids, squalene, α-tocopherol, and Tween 80.

9. The compound adjuvant according to claim 8, characterized in that, The compound adjuvant contains 1-500 μg / mL of cyclic dinucleotide, 0.01-2 mg / mL of cationic lipid, 2-50 mg / mL of squalene, 2-50 mg / mL of α-tocopherol and 1-50 mg / mL of Tween 80.

10. The compound adjuvant according to claim 9, characterized in that, The compound adjuvant contains 1-500 μg / mL c-di-GMP, 0.01-2 mg / mL DOTAP, 2-50 mg / mL squalene, 2-50 mg / mL α-tocopherol, and 1-50 mg / mL Tween 80.

11. The compound adjuvant according to any one of claims 1-10, characterized in that, The human dose of the compound adjuvant is 0.1-1 mL, preferably 0.4-1 mL.

12. The compound adjuvant according to claim 11, characterized in that, The human dose of the compound adjuvant contains: 2-20 mg squalene, 2-20 mg α-tocopherol, 1-20 mg Tween 80, 0.01-1 mg cationic lipids and 1-500 μg cyclic dinucleotides.

13. The compound adjuvant according to claim 12, characterized in that, The human dose of the compound adjuvant contains: 2-20 mg squalene, 2-20 mg α-tocopherol, 1-20 mg Tween 80, 0.01-1 mg DOTAP and 1-500 μg c-di-GMP.

14. The compound adjuvant according to any one of claims 1-13, characterized in that, The compound adjuvant further comprises monophospholipid A.

15. The compound adjuvant according to claim 14, characterized in that, The monophosphoryl lipid A is 3D-MPL.

16. The compound adjuvant according to claim 15, characterized in that, The content of 3D-MPL is 1-500 μg / mL.

17. The compound adjuvant according to claim 15, wherein the human dose of the compound adjuvant contains 1-500 μg of 3D-MPL.

18. The compound adjuvant according to claim 1, characterized in that, The aqueous phase is a phosphate buffer solution, citrate buffer, Tris-HCl buffer, acetate buffer, or citrate-phosphate buffer.

19. A method for preparing the composite adjuvant according to any one of claims 1-18, characterized in that, The method includes mixing an oil phase containing cationic lipids and metabolizable oils with an aqueous phase containing surfactants, emulsifying and homogenizing the mixture to form a cationic emulsion, and then mixing the cationic emulsion with a cyclic dinucleotide to obtain the composite adjuvant.

20. A method for preparing the composite adjuvant according to any one of claims 1-18, characterized in that, The composite adjuvant is obtained by mixing an oil phase containing cationic lipids and metabolizable oils with an aqueous phase containing surfactants and cyclic dinucleotides, emulsifying the mixture, and then homogenizing it.

21. The method according to claim 19 or 20, characterized in that, The oil phase also contains monophospholipid A.

22. Use of the compound adjuvant according to any one of claims 1-18 in the preparation of a medicament for the prevention or treatment of disease.

23. A vaccine composition, characterized in that, It comprises the compound adjuvant as described in any one of claims 1-18 and at least one antigen.

24. The vaccine composition according to claim 23, characterized in that, The antigen is one or more antigens derived from viruses, bacteria, fungi, parasites, or tumors.

25. The vaccine composition according to claim 24, characterized in that, The antigen is derived from at least one of the following: human papillomavirus (HPV), enterovirus that causes hand-foot-mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, novel coronavirus (SARS-CoV-2), hepatitis virus, rabies virus, and metapneumovirus.

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