CN118161605A

CN118161605A - Composite adjuvant containing TLR3 agonist and preparation method and application thereof

Info

Publication number: CN118161605A
Application number: CN202311683005.4A
Authority: CN
Inventors: 袁楚晓; 吴双; 王维龙; 姚文荣; 刘娟; 班靖洋; 洪坤学; 刘勇
Original assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Current assignee: Abzymo Biosciences Co ltd; Jiangsu Ruike Biotechnology Co ltd
Priority date: 2022-12-08
Filing date: 2023-12-08
Publication date: 2024-06-11

Abstract

The invention relates to a compound adjuvant comprising a TLR3 agonist, which comprises an oil-in-water emulsion and the TLR3 agonist, wherein the oil-in-water emulsion comprises squalene, alpha-tocopherol and Tween 80, and the TLR3 agonist is at least one selected from the group consisting of PolyI: C, polyICLC and PolyI: C ₁₂ U. The raw materials of the composite adjuvant component are easy to obtain, the preparation process is simple, and the cost is low; the invention adopts PolyI:C and oil-in-water emulsion as the composite adjuvant, has stable property and small side effect, and can improve the immune effect.

Description

Composite adjuvant containing TLR3 agonist and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical engineering, and in particular relates to a compound adjuvant containing a TLR3 agonist, a preparation method thereof and application thereof in the field of immunotherapy and prevention.

Background

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 recognize structurally conserved molecules produced by microorganisms (bacteria, viruses, parasites, etc.). Once these microorganisms break through physical barriers such as skin or intestinal mucosa, they are recognized by TLRs, which in turn activate immune cell responses. The immune system has the ability to widely recognize pathogenic microorganisms, in part due to the broad presence of Toll-like immune receptors. There are at least 10 different TLRs in mammals.

TLR3 is a member of the TLR family, capable of mediating the transcriptional induction of type 1 interferons (IFN- α/β), pro-inflammatory cytokines (IL-6, IL-10) and chemokines, thus together establishing an antiviral response in the host.

Double stranded RNA analogs (e.g., polyinosinic acid (Poly (I: C)), poly ICLC and Poly I: C ₁₂ U, etc.) are ligands for TLR3 that are capable of being recognized by TLR3 and activating TLR3.TLR3 recognizes Poly (I: C) intracellularly and is activated by it, and is able to recruit downstream adaptor proteins MyD88 and tif, TLR3 induces the expression of inflammatory cytokines such as IL-1, TNF- α, IL-6 and IL-12 via MyD 88-dependent pathways, and is involved in nonspecific antiviral responses, while inducing the expression of costimulatory molecules CD80 and CD86 and antiviral cytokines such as IFN- β, IP-10 via MyD 88-independent pathways, and is involved in inducing differentiation maturation of DCs and antiviral immune responses. In vivo, polyinosinic-acid-assisted vaccines can induce potent cytotoxic T cell immune responses.

In the prior art, liposome adjuvant containing poly inosinic acid is provided, however, the preparation process of the liposome is complex, the cost is high, the encapsulation rate of the liposome on different substances is very different, and the encapsulation rate of the liposome on some antigens is very low, so that the immune effect is affected.

Disclosure of Invention

The invention aims to provide a compound adjuvant containing a TLR3 agonist, which can reduce the production cost and enhance the immune effect of a vaccine.

To achieve the above object, in one aspect, the present invention provides a compound adjuvant comprising a TLR3 agonist, comprising an oil-in-water emulsion and a TLR3 agonist, the TLR3 agonist being selected from at least one of Poly I: C, poly ICLC and PolyI: C ₁₂ U.

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

In some embodiments, the metabolizable oil is squalene.

In some embodiments, 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.

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 squalene, alpha tocopherol, and tween 80.

In some embodiments, the oil-in-water emulsion comprises 2-10wt.% squalene, 2-10wt.% alpha tocopherol, and 0.3-3wt.% tween 80.

In some embodiments, the oil-in-water emulsion comprises squalene, span 85, and tween 80.

In some embodiments, the oil-in-water emulsion comprises 2-10% squalene, 0.2-1.0% tween 80, and 0.1-1.0% span 85 by volume.

In some embodiments, the TLR3 agonist is present in the co-adjuvant in an amount of 0.1-5mg/mL, e.g., 0.1-3mg/mL, preferably 0.2-2mg/mL.

In some embodiments, the TLR3 agonist is Poly I: C, having a molecular weight between 66,000 and 1,200,000 daltons, particularly between 400,000 and 900,000 daltons.

In some embodiments, the human dose of the compound adjuvant comprises: 5-15mg squalene, 5-15mg alpha-tocopherol, 1-10mg tween 80 and 0.05-2.5mg Poly I: C; preferably 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80 and 0.05mg Poly I: C.

In some embodiments, the human dose of the compound adjuvant comprises: 5-15mg squalene, 1-10mg span 85, 1-10mg tween 80 and 0.02-2.5mg Poly I:C; preferably 9.75mg squalene, 1.175mg span 85, 1.175mg Tween 80 and 0.05mg Poly I: C.

In another aspect, the invention provides a method of preparing the compound adjuvant comprising mixing an oil phase with an aqueous phase comprising a TLR3 agonist and an emulsifier, emulsifying and homogenizing.

In some embodiments, the aqueous phase is a phosphate buffer, citrate buffer, tris-HCl buffer, acetate buffer, or citric acid-phosphate buffer.

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

In yet another aspect, the invention also provides the use of the compound adjuvant in the manufacture of a medicament for the prevention or treatment of a disease.

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

In some embodiments, the antigen is one or more antigens derived from a virus, bacterium, fungus, parasite, or tumor.

In some embodiments, the antigen is derived from at least one of Human Papilloma Virus (HPV), enterovirus that causes hand-foot-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.

In some embodiments, the antigen comprises an L1 protein and/or an L2 protein of one or more of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.

In some embodiments, the antigen is derived from one or more of H1N1, H3N2, B/V, and B/Y strains of influenza virus.

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

the raw materials of each component in the composite adjuvant are easy to obtain, the preparation process is simple, and the cost is low; the invention adopts the composite adjuvant of the Poly I, C and the oil-in-water emulsion, has stable property and small side effect, and the Poly I, C and the oil-in-water emulsion play a synergistic effect and can enhance the immune effect.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.

In the description of the present invention, reference to "one embodiment" means that a particular parameter, step, etc. described in the embodiment is at least included in one embodiment according to the present invention. Thus, references to "one embodiment according to the present invention," "in an embodiment," and the like, in this specification are not intended to specify the presence of stated features but rather are intended to be included in particular embodiments, if they are used in the same sense. It will be appreciated by those of skill in the art that the specific parameters, steps, etc. disclosed in one or more of the embodiments of the invention can be combined in any suitable manner.

The invention provides a compound adjuvant comprising a TLR3 agonist, which comprises an oil-in-water emulsion and the TLR3 agonist, wherein the TLR3 agonist is at least one selected from the group consisting of Poly I: C, poly ICLC and Poly I: C ₁₂ U.

TLR agonists

The TLR3 agonist in the invention is polyinosinic acid-polycytidylic acid (PolyI: C) or derivatives thereof. Poly I: C is a double-stranded RNA analogue, consists of a Poly (I) chain and a Poly (C) chain, can simulate dsRNA formed after virus infection, stimulates the organism to produce antiviral immune response and inflammatory response, and has good antiviral effect. It is found that when PolyI: C is directly used as a medicine in clinic, a certain toxicity is generated to the body. In order to reduce the toxicity of the polysaccharide to the organism and improve the capacity of the polysaccharide I/C to stimulate the organism to produce interferon, researchers modify the polysaccharide I/C derivative to create various polysaccharide I/C derivatives.

The complex formed by mixing PolyI:C with Poly L-lysine and dissolving in carboxymethyl cellulose is called Poly-ICLC. Studies have shown that Poly-ICLC can increase the highest concentration of induced IFN in mice by a factor of 5-8 relative to Poly I: C. Poly-ICLC is also toxic to the body. The side effects of the liposome coated Poly-ICLC can be significantly reduced after the liposome is coated.

Poly I: C ₁₂ U is a mismatched double stranded RNA that is capable of up-regulating or down-regulating the 2, 5-ATPase/RNaseL (2, 5-A 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 having a molecular weight between 66,000 and 1,200,000 daltons, e.g., between 75,000 and 1,100,000 daltons, between 96,000 and 950,000 daltons, between 200,000 and 800,000 daltons, between 150,000 and 550,000 daltons, and particularly between 400,000 and 900,000 daltons.

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 metabolisable oil, preferably squalene. In a preferred embodiment, the oil phase further comprises alpha-tocopherol.

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

The buffer solution is phosphate buffer solution, citrate buffer solution, tris-HCl buffer solution, acetate buffer solution or citric acid-phosphoric acid buffer solution.

In some embodiments, the emulsifier comprises one or both of tween 80 and span 85. In some embodiments, the oil-in-water emulsion comprises squalene, alpha tocopherol, and tween 80. The emulsion may comprise a phosphate buffer. These emulsions may contain 2-10wt.% squalene, 2-10wt.% alpha-tocopherol, and 0.3-3wt.% tween 80, and squalene: the weight ratio of alpha-tocopherol is preferably <1 (e.g., 0.90), which may provide a more stable emulsion. 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 preferred embodiments, the weight ratio of the three components (squalene, alpha tocopherol, tween 80) is about 55:61:25, e.g. 1069:1186:486. In some embodiments, the oil-in-water emulsion may be used as an adjuvant for a human vaccine, wherein each human dose comprises 5-15mg squalene, 5-15mg alpha tocopherol, 1-10mg tween 80, preferably in a weight ratio of about 55:61:25, e.g. 1069:1186:486.

In some embodiments, the oil-in-water emulsion comprises squalene, span 85, and tween 80. The emulsion may comprise citrate ions, for example 10mM sodium citrate buffer. In some embodiments, the composition of the emulsion may be 2-10% squalene, 0.2-1.0% tween 80, and 0.1-1.0% span 85 by volume. In some embodiments, the composition of the emulsion is about 5% squalene, about 0.5% tween 80, and about 0.5% span 85 by volume. The composition of the emulsion was 4.3% squalene, 0.5% tween 80 and 0.48% span 85 by weight.

Vaccine composition

Antigens derived from Human Papilloma Virus (HPV) are L1 proteins and/or L2 proteins of each type of HPV. In embodiments of the invention, HPV may be a low-risk HPV (e.g., HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89), a medium-risk HPV (e.g., HPV26, 53, 66, 73, 82), or a 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 and/or L2 proteins 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 and/or L2 proteins 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 proteins and/or L2 proteins 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 causing hand-foot-and-mouth disease mainly comprise Coxsackie group A type 4, 5 type, 6 type, 7 type, 9 type, 10 type 16 type and the like, group B type 2, 5 type and 13 type, enterovirus 71 type (EV 71) and the like. In embodiments of the invention, antigens derived from these enteroviruses may be from one or any combination of the above types, preferably virus-like particles (VLPs) consisting of VP1 protein, VP2 protein, VP3 protein and VP4 protein. VP1 protein, VP2 protein, VP3 protein and VP4 protein are produced by decomposing precursor protein P1 under the action of 3CD protease.

In some embodiments, the antigens derived from these enteroviruses comprise one or more of EV71, coxsackie group a type 6, 10, and 16 viral particles. In a preferred embodiment, the antigens derived from these enteroviruses comprise EV71, coxsackie group a type 6, type 10 and type 16 viral particles.

The protein family with strong immunogenicity of mycobacterium tuberculosis mainly comprises Esx family proteins, PE/PPE family proteins and dosR family proteins. Preferably, the Esx family protein comprises ESAT-6, CFP-10, TB9.8, TB10.3, TB10.4, TB11.0, TB12.9, and the like. The PE/PPE family proteins preferably comprise PPE17, PPE18, PPE34, PPE42, PPE57PE-PGRS33, PE35-PPE68, PE-PGRS62, PE-PGRS17, PE-PGRS11, PE25-PPE41, and the like. The DosR family proteins preferably include Rv2626c, rv2029c, rv2031c, rv2627c, rv3133c, and the like.

In a preferred embodiment of the invention, the antigen derived from tubercle bacillus 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 proteins are PE35 and PPE68, and the dosR family protein is selected from one of Rv2626c, rv2627c, and Rv2031 c. In a preferred embodiment, the antigen derived from tubercle bacillus comprises CFP-10 protein, PE35 protein, PPE68 protein and Rv2627c protein. In a preferred embodiment, the antigen derived from tubercle bacillus comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein and Rv2627c protein. In a preferred embodiment, the antigen derived from tubercle bacillus comprises CFP-10 protein, PE35 protein, PPE68 protein and Rv2626c protein. In a preferred embodiment, the antigen derived from tubercle bacillus comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein and Rv2626c protein. In a preferred embodiment, the antigen derived from tubercle bacillus comprises CFP-10 protein, PE35 protein, PPE68 protein and Rv2031c protein. In a preferred embodiment, the antigen derived from tubercle bacillus comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein and Rv2031c protein.

The antigen derived from Herpes Simplex Virus (HSV) may be gB, gC, gD, gH, gL, gI, ICP0, ICP4, etc. derived from HSV-1 and/or HSV-2. In an embodiment of the invention, the antigen derived from Herpes Simplex Virus (HSV) comprises an 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 a 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 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 a fusion loop domain of an HSV-1gB protein and a fusion loop domain of an HSV-2gB protein.

Antigens derived from Varicella Zoster Virus (VZV) include gB, gC, gE, gH, gI, gK, gL protein of VZV virus, and the like. In a preferred embodiment, the antigen derived from Varicella Zoster Virus (VZV) comprises a truncated gE protein which lacks the carboxy terminal hydrophobic anchor region of the gE protein.

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

Antigens derived from the novel coronavirus (SARS-CoV-2) comprise the SARS-CoV-2 spike protein (S protein), the Receptor Binding Domain (RBD) of the spike protein, or functionally active fragments thereof. In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) is a fusion protein of the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof with the N-terminal domain (NTD) or a functionally active fragment thereof. In a preferred embodiment, the fusion protein further comprises a foldon domain, an Fc domain of a 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 a Receptor Binding Domain (RBD) or a functionally active fragment thereof, an N-terminal domain (NTD) and a 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 immunodominant strain comprising at least one of a prototype strain and a Beta strain and a fusion protein derived from an epidemic dominant strain comprising at least one of a Delta strain and an omacron strain.

In some embodiments, the antigen derived from a novel coronavirus (SARS-CoV-2) comprises a fusion protein formed from an S protein receptor binding region or functionally active fragment thereof derived from an immunodominant strain comprising at least one of a prototype strain and a Beta strain and an S protein receptor binding region or functionally active fragment thereof derived from an immunodominant strain comprising at least one of a Delta strain and an Omicron strain. The Omicron strains include ba.1, ba.2, ba.3, ba.4 and ba.5 variants.

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

Antigens derived from rabies virus include inactivated rabies virus or recombinant proteins 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 a Complex adjuvant

Squalene and alpha-tocopherol are mixed, then phosphate buffer containing 5wt.% of Tween 80 and Poly I: C with molecular weight of 66,000 daltons is added, and after mixing, stirring is carried out at 12000rpm for 20min for emulsification, and homogenization is carried out under 50MPa-100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the composite adjuvant. Each 0.25ml of the compound adjuvant contained 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80 and 0.05mg Poly I: C.

Example 2 preparation of a Complex adjuvant

Mixing squalene and span 85, adding citric acid buffer solution containing Tween 80 and PolyI:C with molecular weight of 66,000 daltons, and shearing at 10000rpm for 15 min.

Homogenizing at 40deg.C under 50-120 MPa until the particle size is 150-170 nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the composite adjuvant. Each 0.25ml of the compound adjuvant contained 9.75mg squalene, 1.175mg span 85, 1.175mg Tween 80 and 0.05mg Poly I: C.

Comparative example 1 preparation of oil-in-water emulsion

Squalene and alpha-tocopherol are mixed, and then phosphate buffer containing 5wt.% tween 80 is added to the mixture, and after mixing, the mixture is stirred at 12000rpm for 20min for emulsification, and homogenized under 50MPa-100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the oil-in-water emulsion. Each 0.25ml of oil-in-water emulsion contained 10.69mg squalene, 11.86mg alpha tocopherol and 4.86mg tween 80.

Comparative example 2 preparation of oil-in-water emulsion

Mixing squalene and span 85, adding citric acid buffer solution containing Tween 80, and shearing at 10000rpm for 15min to obtain colostrum.

Homogenizing at 40deg.C under 50-120 MPa until the particle size is 150-170 nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the oil-in-water emulsion. Each 0.25ml of oil-in-water emulsion contained 9.75mg squalene, 1.175mg span 85 and 1.175mg tween 80.

Comparative example 3 preparation of a Complex adjuvant

Squalene and alpha-tocopherol are mixed, and then phosphate buffer containing 5wt.% tween 80 is added to the mixture, and after mixing, the mixture is stirred at 12000rpm for 20min for emulsification, and homogenized under 50MPa-100MPa until the particle size is 150nm-170nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the oil-in-water emulsion. The oil-in-water emulsion was mixed with a phosphate buffer containing Poly I: C having a molecular weight of 66,000 daltons to give a composite adjuvant. Each 0.25ml of the compound adjuvant contained 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80 and 0.05mg Poly I: C.

Comparative example 4 preparation of a Complex adjuvant

Homogenizing at 20deg.C under 50-120 MPa until particle diameter is 150-170 nm. Finally, the emulsion is filtered by a 0.22 mu m filter to prepare the oil-in-water emulsion. The oil-in-water emulsion was mixed with a citrate buffer containing Poly I: C having a molecular weight of 66,000 daltons to obtain a composite adjuvant. Each 0.25ml of the compound adjuvant contained 9.75mg squalene, 1.175mg span 85, 1.175mg Tween 80 and 0.05mg Poly I: C.

EXAMPLE 3 preparation of HPV vaccine compositions

The complex adjuvants of example 1 and comparative example 3 were mixed with the L1 protein of HPV 45 to obtain vaccine compositions. The dosage (HD) for human is 1.0mL, and the main components comprise: 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80, 0.05mg Poly I:C and 20. Mu.g HPV 45L1 protein. Refrigerating at 2-8 deg.c and storing in dark.

The complex adjuvants of example 2 and comparative example 4 were mixed with the L1 protein of HPV 45 to obtain vaccine compositions. The dosage (HD) for human is 1.0mL, and the main components comprise: 9.75mg squalene, 1.175mg span 85, 1.175mg Tween 80, 0.05mg Poly I:C and 20. Mu.g HPV 45L1 protein. Refrigerating at 2-8 deg.c and storing in dark.

The oil-in-water emulsion of comparative example 1 was mixed with the L1 protein of HPV 45 to obtain a vaccine composition. The dosage (HD) for human is 1.0mL, and the main components comprise: 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg tween 80 and 0.1mg HPV 45L1 protein. Refrigerating at 2-8 deg.c and storing in dark.

The oil-in-water emulsion of comparative example 2 was mixed with the L1 protein of HPV 45 to obtain a vaccine composition. The dosage (HD) for human is 1.0mL, and the main components comprise: 9.75mg squalene, 1.175mg span 85, 1.175mg tween 80 and 20 μg HPV 45L1 protein. Refrigerating at 2-8 deg.c and storing in dark.

Mixing the PolyI:C solution with L1 protein of HPV 45 to obtain vaccine composition. The dosage (HD) for human is 1.0mL, and the main components comprise: 20 μg of L1 protein of HPV 45 and 0.5mg of Poly I: C. Refrigerating at 2-8 deg.c and storing in dark.

After 4 weeks of standing, the vaccine compositions prepared using the compound adjuvants of comparative examples 3 and 4 were found to show delamination, probably due to poor stability of the system when the oil-in-water emulsion was simply mixed with the PolyI: C solution, whereas the stability of the system was higher when the oil-in-water emulsion was prepared using the aqueous phase containing PolyI: C, the PolyI: C was integrated with the oil-in-water emulsion.

Example 4 detection of mouse serum neutralizing antibodies

This example examined the immunogenicity of a novel adjuvant prepared with the addition of a PolyI: C immunostimulant in combination with HPV type 45 antigen, based on an oil-in-water emulsion adjuvant. BALB/c mice were immunized with the adjuvant in combination with HPV45 antigen, respectively, with a muscle immunization, HPV45 antigen immunization dose of 2. Mu.g/mouse, 1/10 human dose was used as adjuvant, and the immunization volume was 100. Mu.l. Serum was isolated by HPV pseudovirus-based methods using a2 week-interval immunization protocol, two weeks after each immunization, and the neutralizing antibody titers and geometric mean titers GMT for each of the 10 serum samples of the first immunization are shown in table 1, and the neutralizing antibody titers and geometric mean titers GMT for each of the 10 serum samples of the second immunization are shown in table 2.

TABLE 1

TABLE 2

Table 1 the results show that after the first immunization, when using the oil-in-water emulsion alone as adjuvant, the geometric mean titer GMT was 256 and 1100, respectively; after the addition of Poly I: C in the oil-in-water emulsion, the geometric mean titre GMT increased to 585 and 2137, respectively, 2.3 and 1.9 times that of the oil-in-water emulsion adjuvant alone. Table 2 the results show that after the second immunization, when using the oil-in-water emulsion alone as adjuvant, the geometric mean titers GMT were 4077 and 10786, respectively; when Poly I: C was added to the oil-in-water emulsion, the geometric mean titre GMT increased to 14998 and 34322, respectively, 3.7 and 3.2 times that of the oil-in-water emulsion adjuvant alone. It can be seen that the oil-in-water emulsion has a synergistic effect with Poly I: C, the complex adjuvant of the present invention can induce an antigen to produce a stronger immune response, and that the geometric mean titre GMT when an oil-in-water emulsion comprising squalene, alpha-tocopherol and Tween 80 is employed is significantly that when an oil-in-water emulsion comprising squalene, span 85 and Tween 80 is employed.

Example 5 Effect of Poly I:C content and molecular weight on immune Effect

In the embodiment, the influence of the content of Poly I and C and the molecular weight on the immune effect is examined by immunizing BALB/C mice with tetravalent influenza virus split vaccine combined with a compound adjuvant.

The tetravalent influenza virus split vaccine is manufactured by Jiangsu Dik Biotechnology Co., ltd, and contains four influenza virus strains of 15 mug of A1 (A/California/7/2009, H1N 1), 15 mug of A3 (A/HongKong/4801/2014, H3N 2), 15 mug of B1 (B/Brisbane/60/2008, B/Victoria, B/V for short) and 15 mug of B2 (B/Phuket/3073/2013, B/Yamagata, B/Y for short) at a dose of 0.5ml per bottle. The composite adjuvant was prepared according to the method of example 1, except that the content of Poly I: C was different from the molecular weight.

Each group of vaccine doses (1/10 HD, i.e., 1.5. Mu.g each of H1N1, H3N2, B/V and B/Y), polyI: C molecular weight, polyI: C dose and emulsion dose (1/10 HD) are shown in Table 3, 6 mice per group were immunized by one-needle immunization procedure, intramuscular injection, 100. Mu.l of immunization volume, blood was collected 14 days after immunization to isolate serum, and each group of immunization effect was evaluated by detecting the blood coagulation-inhibiting antibody titer in the serum.

TABLE 3 Table 3

The Geometric Mean Titers (GMT) of specific hemagglutination-inhibiting antibodies against the H1N1 antigen are shown in table 4.

TABLE 4 Table 4

Group of	GMT for H1N1
		Gr1	317
Gr2	283
		Gr3	317
Gr4	449
		Gr5	317
Gr6	283
		Gr13	252
Gr14	252
		Gr15	28

It can be seen that the compound adjuvants prepared with PolyI:C of different molecular weights all had higher GMT than PolyI:C alone or than the emulsion, indicating that the combined use of PolyI:C and emulsion increased the immune effect, with group Gr4 slightly higher than the other groups, indicating that PolyI:C with a maximum molecular weight below 90,000 may have better effect.

In a study with different doses PolyI:C, the invention examined the Geometric Mean Titers (GMT) of specific hemagglutination-inhibiting antibodies against both H1N1 and B/Y antigens, and the results are shown in Table 5.

TABLE 5

The results show that when the dosage of PolyI:C is 25-150 mug, the better immune effect can be obtained.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. A compound adjuvant comprising a TLR3 agonist, comprising an oil-in-water emulsion comprising squalene, alpha-tocopherol, and tween 80, and a TLR3 agonist selected from at least one of Poly I: C, poly ICLC, and Poly I: C ₁₂ U.

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

3. The compound adjuvant according to claim 1, wherein the TLR3 agonist is present in the compound adjuvant in an amount of 0.1-5mg/mL.

4. The compound adjuvant according to claim 1, wherein the TLR3 agonist is PolyI:c, having a molecular weight between 66,000 and 1,200,000 daltons, in particular between 400,000 and 900,000 daltons.

5. A compound adjuvant according to any one of claims 1 to 4, wherein the human dose of the compound adjuvant comprises: 5-15mg squalene, 5-15mg alpha-tocopherol, 1-10mg tween 80 and 0.02-2.5mg Poly I: C; preferably 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80 and 0.05mg PolyI: C.

6. A method of preparing a compound adjuvant according to any one of claims 1 to 5, comprising mixing an oil phase comprising squalene and alpha-tocopherol with an aqueous phase comprising a TLR3 agonist and tween 80, emulsifying and homogenizing.

7. The method of claim 6, further comprising filtering after homogenizing.

8. Use of a compound adjuvant according to any one of claims 1-5 in the manufacture of a medicament for the prevention or treatment of a disease.

9. A vaccine composition comprising a complex adjuvant according to any one of claims 1-5 and at least one antigen.

10. Vaccine composition according to claim 9, characterized in that 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-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.

11. The vaccine composition of claim 10, wherein the antigen comprises L1 protein and/or L2 protein of one or more of HPV type 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59.

12. The vaccine composition of claim 10, wherein the antigen is derived from one or more of H1N1, H3N2, B/V and B/Y strains of influenza virus.