CN118767124A - Composite adjuvant and its preparation method and application - Google Patents

Abstract

The present invention relates to a composite adjuvant, comprising an oil-in-water emulsion and a TLR3 agonist, wherein the oil-in-water emulsion comprises a cationic lipid, and the TLR3 agonist is selected from at least one of Poly I:C, Poly ICLC and Poly I:C ₁₂ U. The raw materials of the composite adjuvant components of the present invention are relatively easy to obtain, the preparation process is simple and the cost is low; the composite adjuvant formed by the oil-in-water emulsion comprising the cationic lipid and the TLR3 agonist of the present invention has stable properties, small side effects, and can improve the immune effect.

Description

Composite adjuvant and its preparation method and application

Technical Field

The present invention belongs to the field of biomedical engineering, and particularly relates to a composite adjuvant and a preparation method thereof, and application thereof in the field of immunotherapy and prevention.

Background Art

Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity and can be expressed in a variety of cells (such as macrophages and dendritic cells). TLRs are monomeric transmembrane non-catalytic receptors that can recognize structurally conserved molecules produced by microorganisms (bacteria, viruses, parasites, etc.). Once these microorganisms break through physical barriers such as the skin or intestinal mucosa, they will be recognized by TLRs and then activate immune cell responses. The reason why the immune system has the ability to widely recognize pathogenic microorganisms is, to some extent, due to the widespread existence of Toll-like immune receptors. There are at least 10 different TLRs in mammals.

TLR3 is a member of the TLR family that can mediate the transcriptional induction of type 1 interferons (IFN-α/β), proinflammatory cytokines (IL-6, IL-10), and chemokines, thereby jointly establishing the host's antiviral response.

Double-stranded RNA analogs (such as polyinosinic acid (poly(I:C)), Poly ICLC and Poly I:C12U, etc.) are ligands of TLR3 and can be recognized and activated by TLR3. After TLR3 recognizes poly(I:C) and is activated by it in the cell, it can recruit downstream adaptor proteins MyD88 and TRIF. TLR3 can induce the expression of inflammatory cytokines such as IL-1, TNF-α, IL-6 and IL-12 through MyD88-dependent pathways, and participate in nonspecific antiviral responses. At the same time, it can induce the expression of co-stimulatory molecules CD80 and CD86 and antiviral cytokines such as IFN-β and IP-10 through MyD88-independent pathways, and participate in the induction of DC differentiation and maturation and antiviral immune responses. In vivo, vaccines assisted by polyinosinic acid can induce strong cytotoxic T cell immune responses.

The prior art provides liposome adjuvants containing polyinosinic-polycytidylic acid, however, the preparation process of liposomes is relatively complicated and the cost is high, and the encapsulation rate of liposomes for different substances varies greatly, and the encapsulation rate for some antigens is very low, thereby affecting the immune effect.

Summary of the invention

The purpose of the present invention is to provide a composite adjuvant which can reduce production costs and enhance the immune effect of vaccines.

To achieve the above object, on the one hand, the present invention provides a composite adjuvant comprising an oil-in-water emulsion and a TLR3 agonist, wherein the oil-in-water emulsion comprises a cationic lipid, and the TLR3 agonist is at least one selected from Poly I:C, Poly ICLC and Poly I:C ₁₂ U.

In some embodiments, the cationic lipid is selected from one or more of DOTAP, DOTMA, DOP-DEDA, DODMA, DMG-PEG2000, DLin-MC3-DMA and DOGS.

In some embodiments, the oil-in-water emulsion further comprises a metabolisable oil and an emulsifier.

In some embodiments, the metabolizable oil is squalene.

In some embodiments, the emulsifier comprises one or more of polyoxyethylene sorbitan fatty acid esters (Tween), sorbitan fatty acid esters (Span), octoxynol-9 (Triton X-100 or polyethylene glycol octylphenyl ether), and lecithin.

In some embodiments, the emulsifier comprises one or both of Tween 80 and Span 85.

In some embodiments, the oil-in-water emulsion further comprises alpha-tocopherol.

In some embodiments, the oil-in-water emulsion comprises DOTAP, squalene, α-tocopherol, and Tween 80.

In some embodiments, the oil-in-water emulsion comprises 0.01-1 wt.% DOTAP, 2-10 wt.% squalene, 2-10 wt.% α-tocopherol, and 0.3-3 wt.% Tween 80.

In some embodiments, the oil-in-water emulsion comprises DOTAP, squalene, Span 85, and Tween 80.

In some embodiments, the oil-in-water emulsion comprises 0.01-1 wt.% DOTAP, 2-10 wt.% squalene, 0.2-1.0 wt.% Tween 80, and 0.1-1.0 wt.% Span 85.

In some embodiments, the content of TLR3 agonist in the composite adjuvant is 0.1-5 mg/mL.

In some embodiments, the human dose of the composite adjuvant comprises: 5-15 mg squalene, 5-15 mg α-tocopherol, 1-10 mg Tween 80, 0.1-1 mg DOTAP and 0.5-2.5 mg Poly I:C; preferably 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP and 0.5 mg Poly I:C.

In some embodiments, the human dose of the composite adjuvant comprises: 5-15 mg squalene, 1-10 mg Span 85, 1-10 mg Tween 80, 0.1-1 mg DOTAP and 0.2-2.5 mg Poly I:C; preferably 9.75 mg squalene, 1.175 mg Span 85, 1.175 mg Tween 80, 0.5 mg DOTAP and 0.5 mg Poly I:C.

In some embodiments, the TLR3 agonist is Poly I:C, and its molecular weight is between 66,000 and 1200,000 Daltons, in particular between 66,000 and 660,000 Daltons.

On the other hand, the present invention provides a method for preparing the composite adjuvant, comprising mixing an oil phase containing a cationic lipid with an aqueous phase containing an emulsifier, emulsifying the mixture and homogenizing the mixture to form an oil-in-water emulsion, and mixing the oil-in-water emulsion with a TLR3 agonist to obtain the composite adjuvant.

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

In some embodiments, the method further comprises filtering after homogenizing.

On the other hand, the present invention also provides another method for preparing the composite adjuvant, comprising mixing an oil phase containing cationic lipids with an aqueous phase containing an emulsifier through a microfluidic device to form an oil-in-water emulsion, and mixing the oil-in-water emulsion with a TLR3 agonist to obtain the composite adjuvant.

In yet another aspect, the present invention also provides use of the composite adjuvant in preparing a medicament for preventing or treating a disease.

In yet another aspect, the present invention also provides a vaccine composition comprising one or more antigens and the composite adjuvant.

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

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

Compared with the prior art, the present invention has the following beneficial effects:

The raw materials of each component in the composite adjuvant of the present invention are easy to obtain, the preparation process is simple and the cost is low; the composite adjuvant formed by the oil-in-water emulsion containing cationic lipids and Poly I:C is stable in nature and has little side effect. Poly I:C and the oil-in-water emulsion containing cationic lipids play a synergistic role and can enhance the immune effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the hemagglutination inhibition antibody titers against H1N1 strains after using different adjuvants in Example 9 of the present invention;

FIG2 shows the hemagglutination inhibition antibody titers against H3N2 strains after using different adjuvants in Example 9 of the present invention;

FIG3 shows the hemagglutination inhibition antibody titers against Victoria strain after using different adjuvants in Example 9 of the present invention;

FIG. 4 shows the hemagglutination inhibition antibody titer against the Yamagata strain after using different adjuvants in Example 9 of the present invention.

DETAILED DESCRIPTION

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the description of the present invention, when "one embodiment" is mentioned, it means that the specific parameters, steps, etc. described in the embodiment are at least included in one embodiment of the present invention. Therefore, in the description of the present invention, if terms such as "according to one embodiment of the present invention" or "in one embodiment" are used, they are not used to specifically refer to the same embodiment, and if terms such as "in another embodiment", "different embodiments of the present invention", or "another embodiment of the present invention" are used, they are not used to specifically refer to that the mentioned features can only be included in specific different embodiments. It should be understood by those skilled in the art that the specific parameters, steps, etc. disclosed in one or more embodiments of the description of the present invention can be combined in any suitable manner.

The present invention provides a composite adjuvant containing a TLR3 agonist, comprising an oil-in-water emulsion and a TLR3 agonist, wherein the oil-in-water emulsion contains a cationic lipid, and the TLR3 agonist is at least one selected from Poly I:C, Poly ICLC and Poly I:C ₁₂ U.

TLR agonists

The TLR3 agonist in the present invention is polyinosinic-polycytidylic acid (Poly I:C) or its derivatives. Poly I:C is a double-stranded RNA analogue, composed of a poly (I) chain and a poly (C) chain, which can simulate the dsRNA formed after viral infection, stimulate the body to produce antiviral immune response and inflammatory response, and has a good antiviral effect. Studies have found that when Poly I:C is directly used as a drug in clinical practice, it will produce certain toxicity to the body. In order to reduce its toxicity to the body and improve the ability of Poly I:C to stimulate the body to produce interferon, researchers have modified it and created a variety of Poly I:C derivatives.

The complex formed by mixing Poly I:C and poly-L-lysine and dissolving them in carboxymethyl cellulose is called Poly-ICLC. Studies have shown that compared with Poly I:C, Poly-ICLC can increase the maximum concentration of induced IFN in mice by 5-8 times. However, Poly-ICLC is also toxic to the body. After Poly-ICLC is coated with liposomes, its side effects can be significantly reduced.

Poly I:C ₁₂ U is a mismatched double-stranded RNA that can up-regulate or down-regulate the 2,5-adenylate synthetase/RNaseL system and the P68 protein kinase system, and this effect is independent of interferon.

In some embodiments, the TLR3 agonist is selected from one or more of Poly I:C, Poly ICLC and Poly I:C ₁₂ U.

In some preferred embodiments, the TLR3 agonist is Poly I:C, and its molecular weight is between 66,000 and 1200,000 Daltons, for example, between 75,000 and 1100,000 Daltons, between 96,000 and 950,000 Daltons, between 150,000 and 550,000 Daltons, and particularly between 66,000 and 660,000 Daltons. The Poly I:C used in the following examples was prepared by the applicant according to the method described in GB1187830A.

Oil-in-water emulsion

The oil-in-water emulsion of the present invention comprises an aqueous phase, an oil phase and an emulsifier. The oil phase comprises a cationic lipid and a metabolizable oil, preferably squalene. In a preferred embodiment, the oil phase further comprises α-tocopherol.

The cationic lipids in the present invention can be selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylpropyl trimethylammonium chloride (DOTMA), dioleoylglycerophosphate-diethylenediamine conjugate (DOP-DEDA), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) or 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), and dioctadecylamide glycyl spermine (DOGS).

In some embodiments, the weight ratio of squalene to α-tocopherol is 0.8-1, such as 0.85-0.95, preferably 0.9.

In some embodiments, the emulsifier comprises one or more of polyoxyethylene sorbitan fatty acid esters (Tween), sorbitan fatty acid esters (Span), octoxynol-9 (Triton X-100 or polyethylene glycol octylphenyl ether), and lecithin.

The buffer solution is a phosphate buffer solution, a citrate buffer solution, a Tris-HCl buffer solution, an acetate buffer solution or a citric acid-phosphate buffer solution.

In some embodiments, the emulsifier comprises one or both of Tween 80 and Span 85. In some embodiments, the water-in-oil emulsion comprises DOTAP, squalene, alpha-tocopherol and Tween 80. The emulsion may comprise a phosphate buffer. These emulsions may comprise 0.01-1wt.% DOTAP, 2-10wt.% squalene, 2-10wt.% alpha-tocopherol and 0.3-3wt.% Tween 80, and the weight ratio of squalene: alpha-tocopherol is preferably <1 (e.g., 0.90), so that a more stable emulsion can be provided. The weight ratio of squalene to Tween 80 is 1.5-3, e.g., 1.8-2.8, preferably 2.0-2.5, e.g., 2.1, 2.2, 2.3 or 2.4. In some embodiments, the oil-in-water emulsion can be used as an adjuvant for human vaccines, wherein each human dose comprises 5-15 mg squalene, 5-15 mg α-tocopherol, 1-10 mg Tween 80 and 0.1-1 mg DOTAP.

In some embodiments, the oil-in-water emulsion comprises DOTAP, squalene, Span 85 and Tween 80. The emulsion may comprise citrate ions, such as 10 mM sodium citrate buffer. In some embodiments, the emulsion may comprise 0.01-1 wt.% DOTAP, 2-10 wt.% squalene, 0.2-1.0 wt.% Tween 80 and 0.1-1.0 wt.% Span 85. In some embodiments, the composition of the emulsion is 0.05 wt.% DOTAP, 4.3 wt.% squalene, 0.5 wt.% Tween 80 and 0.48 wt.% Span 85. In some embodiments, the oil-in-water emulsion can be used as an adjuvant for human vaccines, wherein each human dose comprises 5-15 mg squalene, 1-10 mg Span 85, 1-10 mg Tween 80, 0.1-1 mg DOTAP.

Vaccine composition

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

The antigens derived from human papillomavirus (HPV) are L1 proteins and/or L2 proteins of various types of HPV. In an embodiment of the present invention, HPV can be low-risk HPV (e.g., HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89), medium-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).

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

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

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

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

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

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

Enteroviruses that cause hand, foot and mouth disease mainly include Coxsackie A group 4, 5, 6, 7, 9, 10, 16, etc., group B type 2, 5 and 13, and enterovirus 71 (EV71), etc. In an embodiment 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 protein, VP2 protein, VP3 protein and VP4 protein. VP1 protein, VP2 protein, VP3 protein and VP4 protein are produced by the decomposition of precursor protein P1 under the action of 3CD protease.

In some embodiments, the antigens derived from these enteroviruses include one or more of EV71, Coxsackie A group 6, 10, and 16 virus particles. In a preferred embodiment, the antigens derived from these enteroviruses include EV71, Coxsackie A group 6, 10, and 16 virus particles.

The protein family with strong immunogenicity of Mycobacterium tuberculosis mainly includes Esx family proteins, PE/PPE family proteins and DosR family proteins. Esx family proteins preferably include ESAT-6, CFP-10, TB9.8, TB10.3, TB10.4, TB11.0, TB12.9, etc. PE/PPE family proteins preferably include PPE17, PPE18, PPE34, PPE42,, PPE57PE-PGRS33, PE35-PPE68, PE-PGRS62, PE-PGRS17, PE-PGRS11, PE25-PPE41, etc. DosR family proteins preferably include Rv2626c, Rv2029c, Rv2031c, Rv2627c, Rv3133c, etc.

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 one of 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 a 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 a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein and Rv2031c 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 Rv2031c protein.

The antigen 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 an embodiment of the present invention, the antigen derived from herpes simplex virus (HSV) comprises HSVgB protein or its functional fragment. In a preferred embodiment, the antigen derived from herpes simplex virus (HSV) comprises the extracellular domain of gB protein or its functional fragment. In one embodiment, the functional fragment of the extracellular domain of gB protein comprises the fusion loop (fusion-loop) domain of gB protein. In one embodiment, the extracellular domain of gB protein comprises at least one amino acid mutation, preferably, the amino acid mutation is 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.

The antigen derived from varicella zoster virus (VZV) comprises VZV virus gB, gC, gE, gH, gI, gK, gL protein, etc. In a preferred embodiment, the antigen derived from varicella zoster virus (VZV) comprises a truncated gE protein lacking the carboxyl terminal hydrophobic anchor region of the gE protein.

The antigen derived from influenza virus comprises inactivated influenza virus, hemagglutinin (HA protein) or neuraminidase (NA protein) of influenza virus. In a preferred embodiment, the antigen derived from influenza virus comprises hemagglutinin (HA protein) of influenza virus. In a preferred embodiment, the antigen derived from influenza virus comprises inactivated influenza virus.

The antigen derived from the novel coronavirus (SARS-CoV-2) comprises the SARS-CoV-2 spike protein (S protein), the receptor binding domain (RBD) of the spike protein, or its functionally active fragment. In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) is a fusion protein formed by the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or its functionally active fragment and the N-terminal domain (NTD) or its functionally active fragment. In a preferred embodiment, the fusion protein further comprises a foldon domain, an Fc domain of a human immunoglobulin, or its functionally active fragment. 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 a receptor binding domain (RBD) or its functionally active fragment, an N-terminal domain (NTD) and a foldon domain or its functionally active fragment. In some embodiments, the antigen derived from the new coronavirus (SARS-CoV-2) comprises a fusion protein derived from an immunodominant strain and a fusion protein derived from a prevalent dominant strain, wherein 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.

In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises a fusion protein formed by a S protein receptor binding region or a functionally active fragment thereof derived from an immunodominant strain and a S protein receptor binding region or a functionally active fragment thereof derived from an epidemic dominant strain, wherein the immunodominant 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. The Omicron strain includes BA.1, BA.2, BA.3, BA.4, and BA.5 variants.

Human hepatitis viruses include hepatitis A, B, C, D, E, and G. In some embodiments, the antigen derived from a hepatitis virus includes hepatitis B surface antigen (HBsAg) derived from hepatitis B.

The antigen derived from rabies virus includes inactivated rabies virus or recombinant protein derived from rabies virus. The recombinant protein is derived from at least one of rabies virus G protein, N protein, M protein, P protein and L protein.

Example 1 Preparation of composite adjuvant

DOTAP, squalene and α-tocopherol are mixed, and then a phosphate buffer containing 5wt.% Tween 80 is added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered using a 0.22μm filter to prepare an oil-in-water emulsion. The oil-in-water emulsion is mixed with a phosphate buffer containing Poly I:C with a molecular weight of 66,000 Daltons to obtain a composite adjuvant. Each 0.5ml human dose of the composite adjuvant contains 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80, 0.5mg DOTAP and 0.5mg Poly I:C.

Example 2 Preparation of composite adjuvant

DOTAP, squalene and Span 85 are mixed evenly, and a citric acid buffer solution containing Tween 80 is added. After mixing evenly, high-speed shearing is performed at 10000rpm for 15 minutes to prepare colostrum, and homogenization is performed at a pressure of 120MPa until the particle size is 150nm-170nm. An oil-in-water emulsion is prepared. The oil-in-water emulsion is mixed with a phosphate buffer containing Poly I: C with a molecular weight of 66,000 Daltons to obtain a composite adjuvant. Each 0.5ml human dose of the composite adjuvant contains 9.75mg squalene, 1.175mg Span 85, 1.175mg Tween 80, 0.5mg DOTAP and 0.5mg Poly I: C.

Example 3 Preparation of composite adjuvant

DOTAP, squalene and Span 85 were mixed evenly as the oil phase, and a citric acid buffer solution containing Tween 80 was used as the aqueous phase. The microfluidic device was operated at a total flow rate of 2 ml/min and a flow rate ratio of oil phase: aqueous phase = 1:4 at a temperature of 20°C to obtain an oil-in-water emulsion. The oil-in-water emulsion was mixed with a phosphate buffer containing Poly I:C with a molecular weight of 66,000 Daltons to obtain a composite adjuvant. Each 0.5 ml human dose of the composite adjuvant contains 9.75 mg squalene, 1.175 mg Span 85, 1.175 mg Tween 80, 0.5 mg DOTAP and 0.5 mg Poly I:C.

Comparative Example 1 Preparation of Oil-in-Water Emulsion

Squalene and α-tocopherol were mixed, and then phosphate buffer containing 5wt.% Tween 80 was added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size was 150nm-170nm. Finally, the emulsion was filtered using a 0.22μm filter to prepare an oil-in-water emulsion. Each 0.5ml human dose of the oil-in-water emulsion contained 10.69mg squalene, 11.86mg α-tocopherol and 4.86mg Tween 80.

Comparative Example 2 Preparation of Oil-in-Water Emulsion

Squalene, α-tocopherol and 3D-MPL were mixed, and then phosphate buffer containing 5wt.% Tween 80 was added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size was 150nm-170nm. Finally, the emulsion was filtered using a 0.22μm filter to prepare an oil-in-water emulsion. Each 0.5ml human dose of the oil-in-water emulsion contained 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80 and 50μg 3D-MPL.

Comparative Example 3 Preparation of composite adjuvant

Squalene and α-tocopherol are mixed, and then a phosphate buffer containing 5wt.% Tween 80 is added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered using a 0.22μm filter to prepare an oil-in-water emulsion. The oil-in-water emulsion is mixed with a phosphate buffer containing Poly I:C with a molecular weight of 66,000 Daltons to obtain a composite adjuvant. Each 0.5ml human dose of the composite adjuvant contains 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80 and 0.5mg Poly I:C.

Comparative Example 4 Preparation of Oil-in-Water Emulsion

Squalene, α-tocopherol and DOTAP were mixed, and then phosphate buffer containing 5wt.% Tween 80 was added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size was 150nm-170nm. Finally, the emulsion was filtered using a 0.22μm filter to prepare an oil-in-water emulsion. Each 0.5ml human dose of the oil-in-water emulsion contained 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80 and 0.5mg DOTAP.

Comparative Example 5 Preparation of Oil-in-Water Emulsion

DOTAP, squalene, 3D-MPL and α-tocopherol were mixed, and then phosphate buffer containing 5wt.% Tween 80 was added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size was 150nm-170nm. Finally, the emulsion was filtered using a 0.22μm filter to prepare an oil-in-water emulsion. Each 0.5ml human dose of the oil-in-water emulsion contained 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80, 0.5mg DOTAP and 50μg 3D-MPL.

Comparative Example 6 Preparation of composite adjuvant

DOTAP, squalene, 3D-MPL and α-tocopherol are mixed, and then a phosphate buffer containing 5wt.% Tween 80 is added and mixed, and then emulsified at 12000rpm for 20min, and homogenized at 100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered using a 0.22μm filter to prepare an oil-in-water emulsion. The oil-in-water emulsion is mixed with a phosphate buffer containing Poly I:C with a molecular weight of 66,000 Daltons to obtain a composite adjuvant. Each 0.5ml human dose of the composite adjuvant contains 10.69mg squalene, 11.86mg α-tocopherol, 4.86mg Tween 80, 0.5mg DOTAP, 50μg 3D-MPL and 0.5mg Poly I:C.

Comparative Example 7 Preparation of liposome adjuvant

DOTAP and cholesterol were mixed, and then anhydrous ethanol and isopropanol were added and mixed to form an alcohol phase; then phosphate buffer was used as the aqueous phase, and the alcohol phase and the aqueous phase were mixed at a flow rate ratio of 1:4 and a total flow rate of 5 ml/min, and the waste liquid before and after was discarded to obtain a cationic liposome adjuvant, named CLB (concentrated liposome). Each 0.5 ml human dose of liposome adjuvant contains 5.6 mg DOTAP and 1.4 mg cholesterol.

Comparative Example 8 Preparation of water-soluble adjuvant

Take 300 μl of polyI:C aqueous solution (molecular weight of polyI:C is 66000, content is 10 mg/ml), add 700 μl of sodium chloride injection, mix at 20 rpm for 20 min to obtain a water-soluble adjuvant. Each 0.5 ml human dose of water-soluble adjuvant contains 1.5 mg of PolyI:C.

Comparative Example 9 Preparation of composite adjuvant

DOTAP was mixed with cholesterol, and then anhydrous ethanol and isopropanol were added and mixed to form an alcohol phase; then phosphate buffer was used as the aqueous phase, and the alcohol phase was mixed with the aqueous phase at a flow rate ratio of 1:4 and a total flow rate of 5 ml/min, and the waste liquid before and after was discarded to obtain a cationic liposome adjuvant, named CLB (concentrated liposome); 10 mg/ml polyI:C aqueous solution was added to CLB at a volume ratio of 7:3, and mixed at a speed of 20 rpm for 20 minutes to obtain a composite adjuvant. Each 0.5 ml human dose of the composite adjuvant contains 5.6 mg DOTAP, 1.4 mg cholesterol and 1.5 mg PolyI:C.

Example 4 Preparation of VZV vaccine composition

The composite adjuvant in Example 1 is mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP, 0.5 mg Poly I:C and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in Example 2 is mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 9.75 mg squalene, 1.175 mg Span 85, 1.175 mg Tween 80, 0.5 mg DOTAP, 0.5 mg Poly I:C and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The oil-in-water emulsion in Comparative Example 1 was mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80 and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The oil-in-water emulsion in Comparative Example 2 was mixed with VZV gE to obtain a vaccine composition. The human dose was 1.0 mL, and the main ingredients included: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 50 μg 3D-MPL and 50 μg VZVgE protein. The composition was refrigerated at 2-8°C and kept away from light.

The composite adjuvant in comparative example 3 is mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg Poly I: C and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The oil-in-water emulsion in Comparative Example 4 was mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The oil-in-water emulsion in Comparative Example 5 was mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP, 50 μg 3D-MPL and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in Comparative Example 6 was mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP, 50 μg 3D-MPL, 0.5 mg Poly I:C and 50 μg VZV gE protein. Refrigerate at 2-8°C and keep away from light.

The Poly I:C solution is mixed with VZV gE to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include: 50 μg VZV GE and 0.5 mg Poly I:C. Refrigerate at 2-8°C and keep away from light.

Example 5 Preparation of HPV vaccine composition

Referring to the preparation method of Example 4, the HPV vaccine composition was prepared using the HPV45 L1 protein. The human dose of the HPV vaccine composition contained 40 μg of HPV45 L1 protein.

Example 6 Preparation of influenza vaccine composition

The composite adjuvant in Example 1 was mixed with HA proteins of four influenza strains: H1N1, H3N2, Victoria and Yamagata to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80, 0.5 mg DOTAP and 0.5 mg Poly I:C, and 15 μg of HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in comparative example 1 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol and 4.86 mg Tween 80, and 15 μg of HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in comparative example 3 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80 and 0.5 mg Poly I: C, and 15 μg of HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in comparative example 4 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main ingredients include: 10.69 mg squalene, 11.86 mg α-tocopherol, 4.86 mg Tween 80 and 0.5 mg DOTAP, and 15 μg of HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The liposome adjuvant in Comparative Example 7 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include: 5.6 mg DOTAP and 1.4 mg cholesterol, and 15 μg HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The water-soluble adjuvant in Comparative Example 8 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include 1.5 mg PolyI:C and 15 μg HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

The composite adjuvant in Comparative Example 9 was mixed with HA proteins of four influenza strains, H1N1, H3N2, Victoria and Yamagata, to obtain a vaccine composition. The human dose is 1.0 mL, and the main components include: 5.6 mg DOTAP, 1.4 mg cholesterol and 1.5 mg PolyI:C, and 15 μg of HA protein of each strain. Refrigerate at 2-8°C and keep away from light.

Example 7 Detection of neutralizing antibodies in HPV immune serum

This example investigates the immunogenicity of a novel adjuvant prepared by adding 3D-MPL or Poly I:C immunostimulant to an oil-in-water emulsion adjuvant and combining it with HPV 45 antigen. The adjuvant was combined with HPV45 antigen to immunize BALB/c mice, and the HPV45 antigen immunization dose was 4 μg/mouse. The adjuvant used 1/10 of the human dose, and the immunization volume was 100 μl. An immunization program was adopted with an interval of 2 weeks. Blood was collected two weeks after each immunization to separate serum, and serum neutralizing antibodies were detected by a method based on HPV pseudovirus. The neutralizing antibody titers and geometric mean titers GMT of 10 serum samples in each group after immunization are shown in Table 1.

Table 1

It can be seen that adding 3D-MPL or Poly I:C to the oil-in-water emulsion adjuvant can significantly improve the immune effect of HPV45 antigen. The oil-in-water emulsion can be used as a basic adjuvant to study the immune effect of the composite adjuvant obtained by further adding other immunostimulants.

Example 8 VZV immune cytokine detection

This example investigates the immunogenicity of a novel adjuvant prepared by adding at least one of DOTAP, 3D-MPL and Poly I:C immunostimulants to an oil-in-water emulsion adjuvant and VZV gE antigen. The adjuvant was combined with VZV gE antigen to immunize BALB/c mice, and the VZV gE antigen immunization dose was 5 μg/mouse. The adjuvants were all 1/10 human doses, and the immunization volume was 100 μl. Varicella vaccine was inoculated on day 0, recombinant herpes zoster vaccine was immunized (1/10 HD) on days 39 and 53, and intracellular cytokine detection was performed on day 67 to evaluate the cellular immune effect of the vaccine, and the geometric mean of each cytokine was calculated, as shown in Table 2.

Table 2

Adjuvant IL-2 ⁺ IL-2 ⁺ IFN-γ ⁺ IFN-γ ⁺ total Comparative Example 2 0.31 0.07 0.12 0.51 Comparative Example 4 0.30 — 0.31 0.62 Comparative Example 5 — — — 0.00 Comparative Example 6 0.43 0.43 0.85 1.71 Example 1 0.44 0.56 0.90 1.89

The results in Table 2 show that compared with adding DOTAP or 3D-MPL to the oil-in-water emulsion adjuvant separately, when DOTAP and 3D-MPL were added simultaneously, the geometric mean of each cytokine could not be calculated due to the lack of valid data detected, indicating that when DOTAP and 3D-MPL were added simultaneously to the oil-in-water emulsion, the immune effect would decrease instead. Based on this, we speculate that the immune stimulation of the two may have an antagonistic effect; when DOTAP and Poly I:C were added simultaneously, each cytokine increased significantly, which was much greater than the level of each cytokine when DOTAP was added alone, indicating that the simultaneous use of DOTAP and Poly I:C can significantly improve the immune effect; when 3D-MPL was further added in the presence of DOTAP and Poly I:C, the cytokines showed a slight decrease, again indicating that DOTAP and 3D-MPL may not be suitable for simultaneous use.

Example 9 Influenza vaccine immune effect experiment

This example investigates the immunogenicity of four influenza HA antigens in combination with various adjuvant forms including oil-in-water emulsion adjuvants, liposome adjuvants, and aqueous adjuvants. According to the method provided in Example 6, different adjuvants were combined with four influenza HA antigens to form vaccine compositions, and BALB/c mice were immunized separately by intramuscular immunization. Both antigens and adjuvants were 1/10 human doses, and the immunization volume was 100 μl. One injection of influenza vaccination was performed on day 0, and blood samples were collected on day 28. The titers of four hemagglutination inhibition antibodies were detected and the geometric mean titer (GMT) was calculated. The results are shown in Figures 1 to 4.

The results in the figure show that the hemagglutination inhibition antibody titer levels of the groups without oil-in-water emulsion (Comparative Examples 7 to 9) are generally low, significantly lower than those of the groups containing oil-in-water emulsion (Example 1, Comparative Examples 1, 3, and 4), indicating that the immunostimulatory effects of DOTAP and polyI:C are inferior to those of oil-in-water emulsion, and even the combination of the two cannot bring significant improvement. In the case of oil-in-water emulsion, when DOTAP or polyI:C is added alone (Comparative Examples 3 and 4), the antibody level produced is not superior to that of the oil-in-water emulsion alone (Comparative Example 1), and the antibody level for the Victoria strain is even slightly lower than that of Comparative Example 1. It can be seen that DOTAP and polyI:C cannot help improve the immunostimulatory effect of oil-in-water emulsion when used alone. When DOTAP and polyI:C were added simultaneously to the oil-in-water emulsion (Example 1), the antibody levels against various strains were significantly increased and were significantly higher than those of almost all other groups (except that the antibody level of the Victoria strain was slightly higher than that of the comparative example 1, which failed to produce a significant difference), indicating that DOTAP and polyI:C can produce a synergistic effect and effectively improve the adjuvant activity in the environment of the oil-in-water emulsion and only in the environment of the oil-in-water emulsion.

The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the scope of protection of the present invention.

Claims (20)

1. A compound adjuvant comprising an oil-in-water emulsion comprising a cationic lipid and a TLR3 agonist selected from at least one of PolyI: C, polyICLC and PolyI:c ₁₂ U.

2. The compound adjuvant according to claim 1, wherein the cationic lipid is selected from one or more of DOTAP, DOTMA, DOP-DEDA, DODMA, DMG-PEG2000, DLin-MC3-DMA and DOGS.

3. A compound adjuvant according to claim 1, wherein the oil-in-water emulsion further comprises a metabolisable oil and an emulsifier, preferably the metabolisable oil is squalene.

4. A compound adjuvant according to claim 3, wherein the emulsifier comprises one or more of a polyoxyethylene sorbitan fatty acid ester (tween), a sorbitan fatty acid ester (span), octoxynol-9 (triton X-100 or polyethylene glycol octylphenyl ether) and lecithin.

5. The compound adjuvant according to claim 4, wherein the emulsifier comprises one or both of tween 80 and span 85.

6. A compound adjuvant according to claim 3, wherein the oil-in-water emulsion further comprises alpha-tocopherol.

7. The compound adjuvant of claim 6, wherein the oil-in-water emulsion comprises DOTAP, squalene, alpha-tocopherol, and tween 80.

8. The compound adjuvant according to claim 7, wherein the oil-in-water emulsion comprises 0.01-1wt.% DOTAP, 2-10wt.% squalene, 2-10wt.% alpha-tocopherol, and 0.1-3wt.% tween 80.

9. The compound adjuvant of claim 5, wherein the oil-in-water emulsion comprises DOTAP, squalene, span 85, and tween 80.

10. The compound adjuvant according to claim 9, wherein the oil-in-water emulsion comprises 0.01-1wt.% DOTAP, 2-10wt.% squalene, 0.2-1.0wt.% tween 80 and 0.1-1.0wt.% span 85.

11. A compound adjuvant according to any one of claims 1 to 10 wherein the TLR3 agonist is present in the compound adjuvant in an amount of 0.1-5mg/mL.

12. The compound adjuvant according to claim 11, wherein the human dose of compound adjuvant comprises: 5-15mg squalene, 5-15mg alpha-tocopherol, 1-10mg tween 80, 0.1-1mg DOTAP and 0.2-2.5mg Poly I: C; preferably 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80, 0.5mg DOTAP and 0.5mg Poly I: C.

13. The compound adjuvant according to claim 11, wherein the human dose of compound adjuvant comprises: 5-15mg squalene, 1-10mg span 85, 1-10mg Tween 80, 0.1-1mg DOTAP and 0.2-2.5mg Poly I: C; preferably 9.75mg squalene, 1.175mg span 85, 1.175mg Tween 80, 0.5mg DOTAP and 0.5mg Poly I: C.

14. The compound adjuvant according to claim 12 or 13, wherein the TLR3 agonist is PolyI:c, having a molecular weight between 66,000 and 1200,000 daltons, in particular between 66,000 and 660,000 daltons.

15. A method of preparing a compound adjuvant according to any one of claims 1 to 14, comprising mixing an oil phase comprising a cationic lipid with an aqueous phase comprising an emulsifier, emulsifying and homogenizing to form an oil-in-water emulsion, and mixing the oil-in-water emulsion with a TLR3 agonist to obtain the compound adjuvant.

16. The method of claim 15, further comprising filtering after homogenizing.

17. A method of preparing a compound adjuvant according to any one of claims 1 to 14, comprising mixing an oil phase comprising a cationic lipid with an aqueous phase comprising an emulsifier via a microfluidic device to form an oil-in-water emulsion, and mixing the oil-in-water emulsion with a TLR3 agonist to obtain the compound adjuvant.

18. Use of a compound adjuvant according to any one of claims 1-14 in the manufacture of a medicament for the prevention or treatment of a disease.

19. A vaccine composition comprising the complex adjuvant of any one of claims 1-14 and at least one antigen.

20. The vaccine composition according to claim 19, wherein the antigen is one or more antigens derived from a virus, a bacterium, a fungus, a parasite or a tumor, preferably the antigen is derived from at least one of Human Papilloma Virus (HPV), enterovirus causing hand-foot-and-mouth disease, tuberculous bacillus, herpes Simplex Virus (HSV), cytomegalovirus (CMV), varicella Zoster Virus (VZV), respiratory Syncytial Virus (RSV), influenza virus, novel coronavirus (SARS-CoV-2), hepatitis virus and rabies virus.