Transmucosally administered oil-in-water emulsion

Abstract

An oil-in-water emulsion, comprising an oil phase and a water phase. The water phase comprises: 0.5-100 mg / mL of a PEG lipid or lipid-based substance, and 10-1000 μg / mL of an antigen or 10-10000 μg / mL of a natural protein or 100-10000 μg / mL of an antibody or any combination thereof. The oil phase comprises: 30-1100 μg / mL of a cationic polymer or an ionizable lipid or protein, the pKa of the ionizable lipid or protein being less than 6.8, and 0.3-25 v / v% of a metabolizable lipid based on the volume of the oil-in-water emulsion, wherein the oil-in-water emulsion has the Young's modulus of 20-800 MPa, and the mean droplet size of 50-800 nm. In addition, further provided are an oil-in-water emulsion adjuvant, and a use of a related emulsion and adjuvant.

Description

Oil-in-water emulsions for transmucosal administration

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number CN202410089002.6, filed on January 22, 2024, entitled “Oil-in-water emulsion,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of biomedicine, and specifically to drug delivery, especially to mucosal penetration and delivery. Background Art

[0004] Existing vaccines and drugs fail to achieve the desired immune response. For example, COVID-19 vaccines, including adenovirus-vectored vaccines, RNA vaccines, inactivated or attenuated vaccines, and subunit vaccines, can still infect the respiratory mucosa and spread among the population despite the production of significant binding and neutralizing antibodies in the serum after subcutaneous injection. Therefore, mucosal immunity is needed to prevent mucosal viral invasion and achieve true immune protection. However, mucosal immunity, due to the presence of structures such as mucus and cilia, rapidly clears foreign antigens or drugs, resulting in extremely low immune or therapeutic efficacy. Furthermore, even if a small amount of antigen or drug is retained, the tight junctions between mucosal epithelial cells hinder further delivery. Therefore, for mucosal viruses, including those in the respiratory and vaginal mucosa, a rationally designed vaccine or drug carrier must not only possess efficient drug, antigen, and bioactive ingredient loading efficiency and good biosafety, but also strong mucus stability, prolonged mucosal residence time, and the ability to penetrate the interstitial spaces of the mucosal epithelium to achieve efficient mucosal delivery and mucosal immune responses.

[0005] Regarding the flexible emulsion that passes through the tight junction gaps of cells and is enriched in specific tissues, the patent CN112999154B that has been applied for discloses a preparation method and application of a flexible deformable albumin oil-in-water emulsion. In this system, albumin is used as the aqueous phase, and squalene and the like are used as the oil phase, and an oil-in-water emulsion is prepared by ultrasound. The emulsion in this system has good deformation ability and can pass through the intercellular gaps under the action of interstitial pressure, thereby enriching in specific tissues. Therefore, the oil-in-water emulsion system solves the problem that the tight junction gaps of cells are difficult to pass through. However, the mucosal delivery of this system has not yet been studied. For mucosal delivery, the primary problem to be solved is to avoid the clearance of mucus and cilia, and to extend the residence time.

[0006] Currently available mucosal vaccine delivery systems include cell-based or viral vector-based recombinant protein purification or peptide antibody delivery for the prevention or treatment of influenza, gastroenteritis, Helicobacter pylori infection, or HIV. For example, patent CN114806994A discloses Bacillus subtilis expressing the structural protein VP1 of the SAT2 foot-and-mouth disease virus and its applications. This invention transforms a recombinant plasmid expressing the structural protein VP3 of the SAT2 foot-and-mouth disease virus into Bacillus subtilis WB800N, demonstrating a strong immune response against foot-and-mouth disease virus infection. It can also be used as a mucosal vaccine, generating high levels of antigen-specific IgG antibodies and mucosal sIgA antibodies, as well as cellular immune responses, after oral immunization. However, these patents all overlook the critical issues of mucociliary clearance and retention time.

[0007] Chitosan particles are currently widely used to stabilize emulsions, imparting a positive charge to the emulsion, which can prolong its mucosal residence time or transiently open tight junctions. For example, CN111617241A and CN107625961A provide chitosan nanoparticle mucosal immune adjuvants. These two patents emphasize the extended adhesion time and penetration of chitosan nanoparticles through the mucosal epithelium. However, positively charged chitosan particles cannot be stably present in the mucus and cilia of the epithelial cells, making them highly likely to be cleared or destroyed, significantly reducing their immune or therapeutic effects. Furthermore, CN113679831A discloses an injectable oil-in-water emulsion mucosal vaccine. This injectable oil-in-water emulsion mucosal vaccine comprises colostrum, all-trans retinoic acid, a metal ion compound, and an antigen. This invention incorporates the mucosal adjuvant all-trans retinoic acid into the system, achieving mucosal immunity through injection. From the perspective of immunization, it avoids the difficulties that need to be overcome in mucosal immunization. Combined with the application of COVID-19 vaccine, mucosal immunization can achieve real immune protection compared to injection immunization.

[0008] Currently, the focus in the field is on penetrating tight junctions, but there is no research on mucosal delivery systems that simultaneously meet the three requirements of mucociliary clearance, enhanced mucosal adhesion, and tight junction penetration. Therefore, providing a system designed and optimized to enhance the stability and mucosal adhesion of drugs or vaccines in mucosal tissues based on the needs of vaccine or drug formulations is of great significance for mucosal pathogen infections and mucosal systemic diseases. Summary of the Invention

[0009] On the one hand, the present application provides an oil-in-water emulsion, which includes an oil phase and an aqueous phase, wherein the aqueous phase includes: 0.5-100 mg / mL of PEG lipid or lipid substance, and 10-1000 μg / mL of antigen, or 10-10000 μg / mL of natural protein or 100-10000 μg / mL of antibody, or any combination thereof; and the oil phase includes: 30-1100 μg / mL of cationic polymer or ionizable lipid or positively charged protein, wherein the cationic polymer is preferably a cationic lipid, wherein the pKa of the ionizable lipid or protein is less than 6.8; and metabolizable oil, preferably, the metabolizable oil is 0.3-25 v / v% based on the volume of the oil-in-water emulsion; wherein the Young's modulus of the oil-in-water emulsion is 20-800 MPa; and the average particle size of the droplets in the oil-in-water emulsion is 50-800 nm.

[0010] On the other hand, the present application provides an oil-in-water emulsion, which includes an oil phase and an aqueous phase, wherein the aqueous phase includes: PEG lipids or lipid substances, and antigens, or natural proteins or antibodies, or any combination thereof; and the oil phase includes: cationic polymers or ionizable lipids or positively charged proteins, wherein the cationic polymers are preferably cationic lipids, wherein the pKa of the ionizable lipids or proteins is less than 6.8; and metabolizable fats, preferably, the metabolizable fats are 0.3-25 v / v% based on the volume of the oil-in-water emulsion; wherein the Young's modulus of the oil-in-water emulsion is 20-800 MPa; and the average particle size of the droplets in the oil-in-water emulsion is 50-800 nm.

[0011] In some embodiments, the aqueous phase further comprises mucin.

[0012] In some embodiments, the aqueous phase comprises 1-100 mg / mL of mucin.

[0013] In some embodiments, the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diglyceride-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dodecyloxy-polyethylene glycol (DPG-PEG), dioctyl ... Ethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- and phosphoethanolamine (DPPE), liposomes, LNPs (lipid nanoparticles) and exosomes, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 550-8000, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 1000-5000, for example, 1500-2500.

[0014] In some embodiments, the antibody or antigen or natural protein is selected from one or more of the following: live attenuated virus vaccine antigens such as influenza virus, COVID-19 virus, hand, foot and mouth virus, rotavirus, inactivated virus vaccine antigens, split vaccine antigens, viral recombinant protein antigens, human serum albumin, genetically recombinant or purified antibodies IgG, IgM, IgA, nanobodies, bispecific antibodies, and pharmaceutical proteins (e.g., IFN-r, IL-2).

[0015] In some embodiments, the antigen is selected from one or more of the following: a tumor antigen, a bacterial antigen (eg, a Helicobacter pylori antigen), a fungal antigen, a viral antigen, and a parasitic antigen.

[0016] In some embodiments, the tumor antigen is a human antigen or a non-human antigen.

[0017] In some embodiments, the antigen is selected from one or more of the following: chemically synthesized antigens, antigens purified and isolated from chicken embryo culture, cell culture, carrier body fluids, organs or tissues, antigens expressed by recombinant genes, recombinant subunit vaccines, attenuated vaccines, inactivated vaccines, split vaccines, polysaccharide conjugate vaccines, nucleic acid vaccines and synthetic peptide antigens.

[0018] In some embodiments, the N-terminus or C-terminus of the antigen can be coupled to a fluorescent marker or a lipid chain, wherein the lipid chain includes one or more of a hydrophobic single chain or multi-grafted carbon chain and a block copolymer, for example, the antigen is a palmitic acid-modified synthetic peptide segment.

[0019] In some embodiments, the cationic polymer or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecyl ammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine poly Ethoxylate diquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), octadec-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate) (SM-102), 1,2-dioctadecenyloxy-3-methylammonium propane (chloride, DOTMA), protamine or cationic peptide, PEI and ionizable lipids.

[0020] In some embodiments, the cationic polypeptide is selected from one or more of the following: RALA, KL4, and polypeptide derivatives containing three or more Rs or Ks.

[0021] In some embodiments, the ionizable lipid is selected from one or more of the following: 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, DLin-MC3-DMA, and SM-102.

[0022] In some embodiments, the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E (tocopherol), ethyl oleate, oleic acid, ethyl lactate, dimethicone, isopropyl laurate, and capric triglyceride.

[0023] In some embodiments, the metabolizable oil is selected from one or more of the following: squalene, olive oil, and simethicone.

[0024] In some embodiments, the oil-in-water emulsion further comprises:

[0025] (E) Toll-like receptor agonists, such as agonists of TLR4, 7, 8, 9, 5, monophosphoryl lipid A (MPLA), CpG, R837, flagellin, and their analogs; and / or

[0026] (F) a STING agonist, such as cGAMP; and / or

[0027] (G) manganese adjuvant; and / or

[0028] (H) saponins and their analogs, such as QS-21; and / or

[0029] (I) hydrophobic drugs, such as one or more of paclitaxel, all-trans retinoic acid, anastrozole and doxorubicin; and / or

[0030] (J) Hydrophilic drugs, such as one or more of letrozole, interleukin and interferon.

[0031] In some embodiments, the oil-in-water emulsion does not include:

[0032] (E) Toll-like receptor agonists, such as agonists of TLR4, 7, 8, 9, 5, monophosphoryl lipid A (MPLA), CpG, R837, flagellin, and their analogs; and / or

[0033] (F) a STING agonist, such as cGAMP; and / or

[0034] (G) manganese adjuvant; and / or

[0035] (H) Saponins and their analogs, such as QS-21.

[0036] In some embodiments, the oil-in-water emulsion has a potential of -20 mV to 70 mV.

[0037] In some embodiments, the metabolizable lipid is 1-20 v / v%, such as 3-15%, such as 4-12% v / v%, 5-10% v / v%, based on the volume of the oil-in-water emulsion.

[0038] In some embodiments, the metabolizable lipid is 0.3-3% v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v%, based on the volume of the oil-in-water emulsion.

[0039] In some embodiments, for the oil-in-water emulsion, the PEG lipid is DSPE-PEG or DMG-PEG, for example, DSPE-PEG 5000, DSPE-PEG 1000, DSPE-PEG 2000 or DMG-PEG-2000, wherein the cationic polymer is a cationic lipid, for example, DDAB, DOTAP or DC-CHOL, wherein the antigen is, for example, an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a herpes zoster antigen, a COVID-19 recombinant virus antigen or an influenza virus antigen, wherein the metabolizable oil is vitamin E, squalene or soybean oil, which is 3-15 v / v% based on the volume of the oil-in-water emulsion, or, based on the volume of the oil-in-water emulsion, 0.3-3 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v%.

[0040] In some embodiments, for the oil-in-water emulsion, the oil-in-water emulsion comprises 3-15 v / v%, preferably 4-10% v / v%, such as 5-7% v / v% of squalene or soybean oil, 3-10 mg / mL, such as 4-8 mg / mL of DSPE-PEG, DSPE-PEG is, for example, DSPE-PEG2000, DSPE-PEG1000, or DSPE-PEG5000, 200-800 μg / mL of PEG. mL, for example 300-500 μg / mL of COVID-19 recombinant virus antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 30-1000 μg / mL, for example 50-900 μg / mL, for example 30-450 μg / mL, for example 600-900 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL of DDAB or DOTAP.

[0041] In some embodiments, for the oil-in-water emulsion, the oil-in-water emulsion includes 0.3-3 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v% of squalene or soybean oil, 3-10 mg / mL, for example, 4-8 mg / mL of DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG5000, 200-800 μg / mL, for example, 300-500 μg / mL of COVID-19 recombinant virus antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 30-1000 μg / mL, for example, 50-900 μg / mL, for example, 30-450 μg / mL, for example, 600-900 μg / mL, for example, 60-200 μg / mL, for example, 70-150 μg / mL, for example, 80-120 μg / mL of DDAB or DOTAP.

[0042] On the other hand, the present application provides an immunogenic composition comprising the oil-in-water emulsion according to any one of the above claims and, if necessary, a pharmaceutically acceptable excipient, wherein the immunogenic composition is used as a vaccine.

[0043] On the other hand, the present application provides a pharmaceutical composition comprising the oil-in-water emulsion according to any one of the above claims, and pharmaceutically acceptable excipients as needed.

[0044] On the other hand, the present application provides the use of the oil-in-water emulsion in the preparation of vaccines or drugs, preferably as a drug sustained-release system, drug delivery system or drug carrier.

[0045] In some embodiments, the vaccine or drug is delivered via the mucosa, such as nasal mucosa delivery or pulmonary mucosa delivery, preferably via nasal mucosa administration.

[0046] On the other hand, the present application provides an oil-in-water emulsion adjuvant, which comprises an oil phase and an aqueous phase,

[0047] The aqueous phase comprises:

[0048] PEG lipids or lipid-like substances, preferably at a concentration of 0.5-50 mg / mL, preferably 0.5-20 mg / mL;

[0049] The oil phase comprises:

[0050] a) cationic lipids or ionizable lipids or positively charged proteins, preferably at a concentration of 30-1000 μg / mL;

[0051] b) metabolizable fat, preferably 0.3-20 v / v% based on the volume of the oil-in-water emulsion;

[0052] The Young's modulus of the oil-in-water emulsion adjuvant is 5-350 MPa, preferably 10-100 MPa. Preferably, the average particle size of the emulsion droplets in the oil-in-water emulsion adjuvant is 60-400 nm.

[0053] In some embodiments, for the oil-in-water emulsion adjuvant, the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diglyceride-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dioctyl-polyethylene glycol (DPG ... EG), dodecyloxy-polyethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- and phosphoethanolamine (DPPE), liposomes, LNPs and exosomes, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 550-8000, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 1000-5000, for example, 1500-2500.

[0054] In some embodiments, for the oil-in-water emulsion adjuvant, the cationic polymer or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecyl ammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine polyoxyethylene ether diquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-dicetadecenyloxy-3-methylammonium propane (chloride, DOTMA), DLin-MC3-DMA, SM-102, protamine or cationic polypeptide and PEI.

[0055] In some embodiments, for the oil-in-water emulsion adjuvant, the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, dimethicone, isopropyl laurate and capric triglyceride.

[0056] In some embodiments, for the oil-in-water emulsion adjuvant, the PEG lipid is DSPE-PEG or DMG-PEG, for example, DSPE-PEG 5000, DSPE-PEG 1000, DSPE-PEG 2000 or DMG-PEG-2000, wherein the cationic polymer is a cationic lipid, for example, DDAB, DOTAP or DC-CHOL, wherein the metabolizable oil is vitamin E, squalene or soybean oil, which is 3-15 v / v% based on the volume of the oil-in-water emulsion adjuvant, or, based on the volume of the oil-in-water emulsion adjuvant, 0.3-3 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v%.

[0057] In some embodiments, the oil-in-water emulsion adjuvant includes 0.5-20 v / v% squalene or soybean oil, 0.5-20 mg / mL, DSPE-PEG such as DSPE-PEG2000 or DSPE-PEG5000, and 30-1000 μg / mL of DDAB or DOTAP.

[0058] In some embodiments, the oil-in-water emulsion adjuvant includes 3-15v / v%, preferably 4-10% v / v%, for example 5-7% v / v% squalene or soybean oil, 3-10 mg / mL, for example 4-8 mg / mL DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG5000, 40-500 μg / mL, for example 50-400 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL DDAB or DOTAP.

[0059] In some embodiments, the oil-in-water emulsion adjuvant includes 3-15 v / v%, preferably 4-10% v / v%, for example 5-7% v / v% squalene or soybean oil, 3-10 mg / mL, for example 4-8 mg / mL DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG5000, 30-1000 μg / mL, for example 50-900 μg / mL, for example, 30-450 μg / mL, for example 600-900 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL DDAB or DOTAP.

[0060] In some embodiments, the oil-in-water emulsion adjuvant includes 0.3-3 v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v% squalene or soybean oil, 3-10 mg / mL, such as 4-8 mg / mL DSPE-PEG, DSPE-PEG is such as DSPE-PEG2000 or DSPE-PEG5000, 40-500 μg / mL, such as 50-400 μg / mL, such as 60-200 μg / mL, such as 70-150 μg / mL, such as 80-120 μg / mL DDAB or DOTAP.

[0061] In some embodiments, the oil-in-water emulsion adjuvant includes 0.3-3 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v% squalene or soybean oil, 3-10 mg / mL, for example, 4-8 mg / mL DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG5000, 30-1000 μg / mL, for example, 50-900 μg / mL, for example, 30-450 μg / mL, for example, 600-900 μg / mL, for example, 60-200 μg / mL, for example, 70-150 μg / mL, for example, 80-120 μg / mL DDAB or DOTAP.

[0062] In some embodiments, for the oil-in-water emulsion adjuvant, wherein the mass volume ratio of cationic lipid: squalene or soybean oil is 1-25ug / ul, PEG lipid: preferably, the mass volume ratio of PEG lipid: squalene or soybean oil is 5-450ug / ul, preferably, the oil-in-water emulsion adjuvant contains 0.5-15v / v% squalene or soybean oil, 0.5-12mg / mL of DSPE-PEG, DSPE-PEG is, for example, DSPE-PEG2000 or DSPE-PEG5000, and 30-450μg / mL of DDAB or DOTAP.

[0063] On the other hand, the present application provides an immunogenic composition comprising any of the above-mentioned oil-in-water emulsion adjuvants, and an antigen, and pharmaceutically acceptable excipients as needed, wherein the immunogenic composition is used as a vaccine. Preferably, the concentration of the antigen is 10-1000 μg / mL.

[0064] In some embodiments, for the immunogenic composition, the antigen is a live attenuated virus vaccine antigen, an inactivated virus vaccine antigen, a split vaccine antigen, a virus-like particle vaccine antigen, a subunit vaccine antigen, a polysaccharide conjugate vaccine antigen, a nucleic acid vaccine antigen or a synthetic peptide vaccine antigen, for example, an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a COVID-19 recombinant virus antigen or an influenza virus antigen, an influenza virus split antigen and / or an influenza recombinant protein antigen.

[0065] On the other hand, the present application provides a method for preparing the immunogenic composition, which comprises mixing the oil-in-water emulsion adjuvant of the present invention and a solution containing an antigen, preferably, wherein the volume ratio of the oil-in-water emulsion adjuvant and the antigen-containing solution is 1:20-1:1, preferably 1:10-1:1 or 1:5-1:1.

[0066] On the other hand, the present application provides a pharmaceutical composition comprising the oil-in-water emulsion adjuvant of the present invention and a drug, and pharmaceutically acceptable excipients as needed. Preferably, the drug is, for example, 10-1000 μg / mL of an antigen, 10-10000 μg / mL of a natural protein, or 100-10000 μg / mL of an antibody, or any combination thereof.

[0067] On the other hand, the present application provides the use of the oil-in-water emulsion adjuvant in the preparation of vaccines or drugs, preferably as a drug sustained-release system, drug delivery system or drug carrier.

[0068] In some embodiments, the vaccine or drug is delivered via the mucosa, preferably via the nasal mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 shows the antigen loading efficiency of different schemes (1, 2, 4, 8, 9, 16, 20, 13 and 18) in Table 3.

[0070] Figure 2 shows the emulsions prepared by Schemes 3, 26, and 27 in Table 3, and a comparison of the stabilities of the emulsions in mucus buffer;

[0071] FIG3 is a line graph showing the mucosal residence time of the antigens in the emulsions prepared by different schemes (19, 13, 6, 23, 15, 8, 2, 26 and 27, and antigens as controls) in Table 3.

[0072] Figure 4 is a bar graph of the Young's modulus of the emulsions prepared by different solutions (19, 13, 6, 23, 15, 8, 2, 26 and 27, as well as particles as a control) in Table 3

[0073] Figure 5 is a graph showing the efficiency of emulsion deformation through intercellular spaces prepared by different schemes (1, 4, 8, 13, 17, 21, 2, 26 and 27, as well as particles as controls) in Table 3.

[0074] Figure 6 is a histogram of the particle size of emulsions prepared with different cationic lipids in Table 3

[0075] Figure 7 is a histogram of the particle size of emulsions prepared with different PEG lipids in Table 3

[0076] Figure 8 is a histogram of the particle size of the emulsions prepared with different PEG molecular weight lengths in Table 3

[0077] Figure 9 is a histogram of the particle size of the emulsions prepared with different oil phases in Table 3

[0078] Figure 10 is a histogram of particle size of emulsions prepared with different oil phase concentrations in Table 3

[0079] Figure 11 is a histogram of particle size of emulsions prepared with different mass ratios of component C and component A in Table 3

[0080] Figure 12 shows the evaluation of the mucosal and systemic immune effects of emulsions prepared with different schemes in Table 3 (2, 12, 13, and 4, as well as aluminum adjuvant as a positive control and antigen as a negative control) on nasal immunization with COVID-19 antigens.

[0081] FIG13 is an evaluation of the mucosal and systemic immune effects of emulsions prepared with different regimens (1, 7, 19, and 24, as well as aluminum adjuvant as a positive control and antigen as a negative control) in Table 3 on nasal immunization with influenza antigens.

[0082] FIG14 shows the mucosal immune effects of the lungs of emulsions prepared by different schemes (2, 12, 13 and 4, as well as aluminum adjuvant as a positive control and antigen as a negative control) in Table 3.

[0083] FIG15 is an evaluation of the mucosal immune effect of emulsions prepared with different regimens (5, 8, 17 and 20, as well as aluminum adjuvant as a positive control and antigen as a negative control) in Table 3 on enterovirus antigens.

[0084] Figure 16 is the evaluation of lung viral load in challenged mice after immunization with emulsions prepared with different regimens in Table 3 (1, 7, 19 and 24, as well as marketed vaccines as positive controls and antigen and PBS groups as negative controls).

[0085] FIG17 is an evaluation of the mucosal and systemic immune effects of the emulsion prepared by Scheme 4 in Table 3 on different small molecules.

[0086] FIG18 is an in vivo safety evaluation of the emulsion prepared by Scheme 4 in Table 3.

[0087] FIG19 shows the antigen loading efficiency after different solutions in Table 5 are mixed with antigens.

[0088] FIG20 is a comparison of the stability of solution No. 3 in Table 5 after mixing with antigen and solution No. 4 in Table 3 in mucus buffer;

[0089] FIG21 is a line graph showing the residence time of the antigen in the mucosa after the emulsions prepared by different schemes in Table 5 are mixed with the antigen.

[0090] FIG22 is a graph showing the efficiency of deformation and passage through intercellular spaces of the emulsions prepared by different schemes in Table 5 after being mixed with antigens.

[0091] Figure 23 is the evaluation of the mucosal immune and systemic immune effects of nasal immunization of mice after the emulsions prepared by different schemes in Table 5 (3, 8, 14 and 17, as well as the marketed vaccine as a positive control and the PBS and control group co-ultrasound group as negative controls) were mixed with flow-lytic antigens.

[0092] Figure 24 is the evaluation of the mucosal immune and systemic immune effects of nasal drops immunization of mice after the emulsions prepared with different schemes in Table 5 (3, 8, 14 and 17, as well as the marketed vaccine as a positive control and the PBS and control group ultrasound group as negative controls) were mixed with the new coronavirus Spike protein.

[0093] Figure 25 is the evaluation of lung viral load in mice challenged with emulsions prepared with different schemes in Table 5 (3, 8, 14 and 17, as well as the marketed vaccine as a positive control and the PBS and control group ultrasound group as negative controls) after immunization with influenza A virus split antigen.

[0094] Figure 26 is the evaluation of the lung viral load of mice challenged with emulsions prepared with different schemes in Table 5 (3, 8, 14 and 17, as well as the marketed vaccine as a positive control and the PBS and control group ultrasound group as negative controls) after immunization with influenza B virus split antigen. DETAILED DESCRIPTION

[0095] The "PEG lipid" in this application refers to a compound that combines PEG with lipids. The "PEG lipid" in this application can be, for example, DSPE-PEG-2000, DSPE-PEG5000, DSPE-PEG1000 and DSPE-PEG-liposome.

[0096] The PEG lipid or lipid-like substance in the present application is selected from one or more of the following: 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diglyceride-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dodecyloxy-polyethylene glycol (DPG-PEG), dioctyl ... Alcohol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- and phosphoethanolamine (DPPE), liposomes, LNPs (lipid nanoparticles) and exosomes, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 550-8000, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 1000-5000, for example, 1500-2500.

[0097] "Lipids" as used herein refer to a class of compounds or structures composed of lipid molecules, which are formed by chemical bonds between long-chain fatty acids and other small molecules (such as glycerol and phosphoric acid). Examples of "lipids" used herein include DPPC, DPPE, liposomes, LNPs (lipid nanoparticles), and exosomes.

[0098] "Antigen" in this application refers to all protein substances that can induce an immune response in the body. Antigens can be specifically recognized and bound by antigen receptors on the surface of T or B lymphocytes to produce immune response products, and can specifically bind to corresponding products in vivo and in vitro. Immunogenicity refers to the ability of an antigen to induce a specific immune response in the body, produce antibodies and / or sensitized lymphocytes; immunoreactivity refers to the ability to specifically bind to corresponding immune effector substances in vivo and in vitro. The "antigen" in this application can be, for example, recombinant influenza virus, COVID-19 virus, hand, foot and mouth disease virus, rotavirus and viral recombinant protein antigens, etc.

[0099] "Native proteins" as used herein refer to proteins purified from natural sources. Natural proteins may be modified, such as by precursor protein cleavage, disulfide bond formation, glycosylation, or phosphorylation. "Native proteins" as used herein may include, for example, human serum albumin, pharmaceutical proteins, and mucin.

[0100] "Antibody" as used herein refers to an immunoglobulin that specifically binds to an antigen. Antibodies have binding sites that bind to corresponding antigenic determinants. "Antibodies" as used herein may include, for example, recombinant or purified IgG, IgM, IgA, nanobodies, and bispecific antibodies.

[0101] The "cationic polymer" in this application is a type of high molecular compound with cationic properties, which contains positively charged groups in its molecular structure and can form stable complexes or compounds by interacting with negatively charged substances.

[0102] The "cationic polymer" in the present application may be, for example, didodecyldimethylammonium bromide (DDAB), dodecylammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, octadecylamine polyoxyethylene ether diquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-dioctadecenyloxy-3-methylammonium propane (chloride, DOTMA). The "cationic polymer" in the present application may also be, for example, cationic polyacrylamide (CPAM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), protamine, or cationic polypeptides and PEI.

[0103] "Ionizable lipids" in this application refer to a class of lipid molecules that can carry a positive charge or a negative charge under specific pH conditions. "Ionizable lipids" in this application can be, for example, DLin-MC3-DMA and SM-102.

[0104] "Protein" in this application refers to a nitrogen-containing biological macromolecule with a specific structure formed by many amino acids connected by peptide bonds. "Positively charged protein" in this application can be, for example, protamine.

[0105] "Metabolizable fats" in this application refer to a class of lipid molecules that can be broken down and utilized by metabolic pathways in an organism, typically composed of glycerol and fatty acids, and can be degraded by enzymes in the organism into energy or other metabolites. "Metabolizable fats" in this application can, for example, be squalene, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, simethicone, isopropyl laurate, and capric triglyceride.

[0106] The "cationic polypeptide" in this application is a class of positively charged polypeptide molecules that generally have good cell permeability and intracellular targeting capabilities. The "cationic polypeptide" in this application can be, for example, RALA, KL4, and polypeptide derivatives containing three or more R or K groups.

[0107] In one aspect, the present application provides an oil-in-water emulsion adjuvant comprising an oil phase and an aqueous phase.

[0108] The aqueous phase comprises:

[0109] PEG lipids or lipid-like substances, preferably at a concentration of 0.5-50 mg / mL, for example 0.5-20 mg / mL,

[0110] The oil phase comprises:

[0111] a) cationic lipids or ionizable lipids or positively charged proteins, preferably at a concentration of 30-1000 μg / mL;

[0112] b) metabolizable fat, preferably 0.3-25 v / v% based on the volume of the oil-in-water emulsion;

[0113] The Young's modulus of the oil-in-water emulsion adjuvant is 5-350 MPa, preferably 10-100 MPa. Preferably, the average particle size of the emulsion droplets in the oil-in-water emulsion adjuvant is 100-400 nm.

[0114] In some embodiments, for the oil-in-water emulsion adjuvant, the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diglyceride-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dioctyl-polyethylene glycol (DPG ... EG), dodecyloxy-polyethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- and phosphoethanolamine (DPPE), liposomes, LNPs and exosomes, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 550-8000, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 1000-5000, for example, 1500-2500.

[0115] In some embodiments, for the oil-in-water emulsion adjuvant, the molecular weight of the polyethylene glycol in the PEG lipid is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 4000, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 6000, 6500, 7000, 7500, 8000, or any range between the above values.

[0116] In some embodiments, the concentration of each component is calculated based on the volume of the final emulsion adjuvant.

[0117] In some embodiments, for the oil-in-water emulsion adjuvant, the aqueous phase comprises 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg / mL of PEG lipid or lipid-like substance (component A in Table 5) or any range of PEG lipid or lipid-like substance between the above values.

[0118] In some embodiments, for the oil-in-water emulsion adjuvant, the cationic polymer is a cationic lipid, such as DDAB, DOTAP or DC-CHOL.

[0119] In some embodiments, for the oil-in-water emulsion adjuvant, the cationic polymer or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecyl ammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine polyoxyethylene ether diquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-dicetadecenyloxy-3-methylammonium propane (chloride, DOTMA), DLin-MC3-DMA, SM-102, protamine or cationic polypeptide and PEI.

[0120] In some embodiments, for the oil-in-water emulsion adjuvant, the oil phase comprises 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 940, 950, 960, 970, 980, 990 or 1000 μg / mL of a cationic polymer or an ionizable lipid or protein (component B in Table 5) or any range of cationic polymers or ionizable lipids or proteins between the above values.

[0121] In some embodiments, for the oil-in-water emulsion adjuvant, the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, dimethicone, isopropyl laurate and capric triglyceride.

[0122] In some embodiments, for the oil-in-water emulsion adjuvant, the oil phase comprises 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% (v / v) of metabolizable fat (component D in Table 3) or any range of metabolizable fats therebetween.

[0123] In some embodiments, the metabolizable lipid is 3-15% v / v% based on the volume of the oil-in-water emulsion adjuvant, for example, 4-12% v / v%, 5-10% v / v%.

[0124] In some embodiments, the metabolizable lipid is 0.3-2.5% v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v%, based on the volume of the oil-in-water emulsion.

[0125] In some embodiments, the Young's modulus of the oil-in-water emulsion adjuvant is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 5 ... 700, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 2000, 2010

[0126] In some embodiments, the Young's modulus of the oil-in-water emulsion adjuvant is preferably 5-350 MPa, such as 10-100 MPa, such as 30-70 MPa.

[0127] In some embodiments, the average particle size of the droplets in the oil-in-water emulsion adjuvant is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410 , 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 or 800 nm or any range therebetween.

[0128] In some embodiments, the average particle size of the droplets in the oil-in-water emulsion adjuvant is preferably 80-400 nm, such as 100-300 nm.

[0129] In some embodiments, for the oil-in-water emulsion adjuvant, the PEG lipid is DSPE-PEG or DMG-PEG, for example, DSPE-PEG 5000, DSPE-PEG 2000 or DMG-PEG-2000, wherein the cationic polymer is a cationic lipid, for example, DDAB, DOTAP or DC-CHOL, wherein the metabolizable oil is vitamin E, squalene or soybean oil, which is 3-15 v / v% based on the volume of the oil-in-water emulsion adjuvant, or, based on the volume of the oil-in-water emulsion adjuvant, is 0.3-2.5 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v%.

[0130] In some embodiments, the oil-in-water emulsion adjuvant includes 3-15v / v%, preferably 4-10% v / v%, for example 5-7% v / v% squalene, 3-10 mg / mL, for example 4-8 mg / mL DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG1000, 40-500 μg / mL, for example 50-400 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL DDAB or DOTAP.

[0131] In some embodiments, the oil-in-water emulsion adjuvant includes 0.3-2.5 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v% squalene, 3-10 mg / mL, for example, 4-8 mg / mL DSPE-PEG, DSPE-PEG is for example DSPE-PEG2000 or DSPE-PEG5000, 40-500 μg / mL, for example, 50-400 μg / mL, for example, 60-200 μg / mL, for example, 70-150 μg / mL, for example, 80-120 μg / mL DDAB or DOTAP.

[0132] In some embodiments, the amount of surfactant in the oil-in-water emulsion is 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or any range therebetween.

[0133] In some embodiments, the oil-in-water emulsion contains no or almost no surfactant.

[0134] On the other hand, the present application provides a method for preparing an oil-in-water emulsion, which comprises the following steps: configuring the lipid or lipid-like substance into an aqueous phase, dissolving the cationic polymer or ionizable lipid or positively charged protein in a metabolizable oil to configure it into an oil phase, and then mixing the aqueous phase and the oil phase to prepare the oil-in-water emulsion adjuvant of the present application, and then mixing the oil-in-water emulsion adjuvant of the present application with a solution containing an antigen, preferably, the volume ratio of the oil-in-water emulsion adjuvant and the antigen-containing solution is 1:10-1:1, preferably 1:5-1:1.

[0135] On the other hand, the present application provides an oil-in-water emulsion, which includes an oil phase and an aqueous phase, wherein the aqueous phase includes: 0.5-100 mg / mL of PEG lipid or lipid substance, and 10-1000 μg / mL of antigen, or 10-10000 μg / mL of natural protein or 100-10000 μg / mL of antibody, or any combination thereof; and the oil phase includes: 30-1100 μg / mL of cationic polymer or ionizable lipid or positively charged protein, wherein the cationic polymer is preferably a cationic lipid, wherein the pKa of the ionizable lipid or positively charged protein is less than 6.8; and metabolizable oil, preferably, the metabolizable oil is 0.3-25 v / v% based on the volume of the oil-in-water emulsion; wherein the Young's modulus of the oil-in-water emulsion is 20-800 MPa; and the average particle size of the droplets in the oil-in-water emulsion is 50-800 nm.

[0136] In some embodiments, the concentration of each component is calculated based on the volume of the final emulsion.

[0137] In some embodiments, for the oil-in-water emulsion, the aqueous phase comprises 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 98, 99, or 100 mg / mL of PEG lipid or lipid-like substance (component A in Table 3) or any range therebetween.

[0138] In some embodiments, for the oil-in-water emulsion, the aqueous phase comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 940, 950, 960, 970, 980, 990 or 1000 μg / mL of the antigen (one of the B components in Table 3) or any range therebetween.

[0139] In some embodiments, the antigen is a protein antigen.

[0140] In some embodiments, for the oil-in-water emulsion, the aqueous phase comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 μg / mL of a native protein (one of the B components in Table 3), or any range of native protein therebetween.

[0141] In some embodiments, for the oil-in-water emulsion, the aqueous phase comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 μg / mL of antibody (one of component B in Table 3), or any range of antibodies therebetween.

[0142] In some embodiments, for the oil-in-water emulsion, the oil phase comprises 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580 940, 950, 960, 970, 980, 990, 1000 or 1100 μg / mL of a cationic polymer or an ionizable lipid or protein (component C in Table 3), or any range therebetween.

[0143] In some embodiments, for the oil-in-water emulsion, the oil phase comprises 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% (v / v) metabolizable fat (component D in Table 3 and component C in Table 5), or any range of metabolizable fats and oils therebetween.

[0144] In some embodiments, the metabolizable lipid is 1-20 v / v%, such as 3-15%, such as 4-12% v / v%, 5-10% v / v%, based on the volume of the oil-in-water emulsion.

[0145] In some embodiments, the metabolizable lipid is 0.3-2.5% v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v%, based on the volume of the oil-in-water emulsion.

[0146] In some embodiments, for the oil-in-water emulsion, the pKa of the ionizable lipid or protein is less than 6.8, less than 6.7, less than 6.6, less than 6.5, less than 6.4, less than 6.3, less than 6.2, less than 6.1, less than 6, less than 5.9, less than 5.8, less than 5.7, less than 5.6, less than 5.5, less than 5.4, less than 5.3, less than 5.2, less than 5.1, or less than 5.

[0147] In some embodiments, for the oil-in-water emulsion, the pKa of the ionizable lipid or protein is 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, or any range therebetween.

[0148] In some embodiments, the Young's modulus of the oil-in-water emulsion is 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700 700, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 2000, 2010

[0149] In some embodiments, the Young's modulus of the oil-in-water emulsion is preferably 25-200 MPa, such as 30-100 MPa, such as 30-70 MPa.

[0150] In some embodiments, the average particle size of the droplets in the oil-in-water emulsion is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 700, 790, or 800 nm, or any range therebetween.

[0151] In some embodiments, the average particle size of the droplets in the oil-in-water emulsion is preferably 80-500 nm, for example, 100-300 nm.

[0152] In some embodiments, the surfactant content of the oil-in-water emulsion is less than 2%.

[0153] In some embodiments, the amount of surfactant in the oil-in-water emulsion is less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%.

[0154] In some embodiments, the amount of surfactant in the oil-in-water emulsion is 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or any range therebetween.

[0155] In some embodiments, the oil-in-water emulsion contains no or almost no surfactant.

[0156] In some embodiments, for the oil-in-water emulsion, the aqueous phase further comprises mucin.

[0157] In some embodiments, for the oil-in-water emulsion, the aqueous phase includes 1-100 mg / mL of mucin.

[0158] In some embodiments, for the oil-in-water emulsion, the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diglyceride-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dodecanol-1,2-di ... Alkoxy-polyethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- and phosphoethanolamine (DPPE), liposomes, LNPs (lipid nanoparticles) and exosomes, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 550-8000, preferably, the molecular weight of the polyethylene glycol in the PEG lipid is 1000-5000, for example, 1500-2500.

[0159] In some embodiments, for the oil-in-water emulsion, the molecular weight of the polyethylene glycol in the PEG lipid is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400 700, 7500, 8000, or any range between the above values.

[0160] In some embodiments, the antibody or antigen or natural protein is selected from one or more of the following: live attenuated virus vaccine antigens such as influenza virus, COVID-19 virus, hand, foot and mouth virus, rotavirus, inactivated virus vaccine antigens, split vaccine antigens, viral recombinant protein antigens, human serum albumin, genetically recombinant or purified antibodies IgG, IgM, IgA, nanobodies, bispecific antibodies, and pharmaceutical proteins (e.g., IFN-r, IL-2).

[0161] In some embodiments, the antigen is selected from one or more of the following: a tumor antigen, a bacterial antigen (eg, a Helicobacter pylori antigen), a fungal antigen, a viral antigen, and a parasitic antigen.

[0162] In some embodiments, the tumor antigen is a human antigen or a non-human antigen.

[0163] In some embodiments, the antigen is selected from one or more of the following: chemically synthesized antigens, antigens purified and isolated from chicken embryo culture, cell culture, carrier body fluids, organs or tissues, antigens expressed by recombinant genes, recombinant subunit vaccines, attenuated vaccines, inactivated vaccines, split vaccines, polysaccharide conjugate vaccines, nucleic acid vaccines and synthetic peptide antigens.

[0164] In some embodiments, the N-terminus or C-terminus of the antigen can be coupled to a fluorescent marker or a lipid chain, wherein the lipid chain includes one or more of a hydrophobic single chain or multi-grafted carbon chain and a block copolymer, for example, the antigen is a palmitic acid-modified synthetic peptide segment.

[0165] In some embodiments, the cationic polymer or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecylammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine polyoxyethylene ether diquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-dioctadecenyloxy-3-methylammonium propane (chloride, DOTMA), protamine or cationic polypeptide, PEI and ionizable lipid.

[0166] In some embodiments, the cationic polypeptide is selected from one or more of the following: RALA, KL4, and polypeptide derivatives containing three or more Rs or Ks.

[0167] In some embodiments, the ionizable lipid is selected from one or more of the following: 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, DLin-MC3-DMA, and SM-102.

[0168] In some embodiments, the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E (tocopherol), ethyl oleate, oleic acid, ethyl lactate, dimethicone, isopropyl laurate, and capric triglyceride.

[0169] In some embodiments, the metabolizable oil is selected from one or more of the following: squalene, olive oil, and simethicone.

[0170] In some embodiments, the oil-in-water emulsion further comprises:

[0171] (E) Toll-like receptor agonists, such as agonists of TLR4, 7, 8, 9, 5, monophosphoryl lipid A (MPLA), CpG, R837, flagellin, and their analogs; and / or

[0172] (F) a STING agonist, such as cGAMP; and / or

[0173] (G) manganese adjuvant; and / or

[0174] (H) saponins and their analogs, such as QS-21; and / or

[0175] (I) hydrophobic drugs, such as one or more of paclitaxel, all-trans retinoic acid, anastrozole and doxorubicin; and / or

[0176] (J) Hydrophilic drugs, such as one or more of letrozole, interleukin and interferon.

[0177] In some embodiments, the oil-in-water emulsion does not include:

[0178] (E) Toll-like receptor agonists, such as agonists of TLR4, 7, 8, 9, 5, monophosphoryl lipid A (MPLA), CpG, R837, flagellin, and their analogs; and / or

[0179] (F) a STING agonist, such as cGAMP; and / or

[0180] (G) manganese adjuvant; and / or

[0181] (H) Saponins and their analogs, such as QS-21.

[0182] In some embodiments, the oil-in-water emulsion has a potential of -20 mV to 70 mV.

[0183] In some embodiments, the potential of the oil-in-water emulsion is -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 65, 66, 67, 68, 69 or 70 mV, or any range therebetween.

[0184] In some embodiments, for the oil-in-water emulsion, the PEG lipid is DSPE-PEG or DMG-PEG, for example, DSPE-PEG 5000, DSPE-PEG 2000 or DMG-PEG-2000.

[0185] In some embodiments, for the oil-in-water emulsion, the cationic polymer is a cationic lipid, such as DDAB, DOTAP or DC-CHOL.

[0186] In some embodiments, for the oil-in-water emulsion, the antigen is, for example, an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a COVID-19 recombinant virus antigen, or an influenza virus antigen.

[0187] In some embodiments, for the oil-in-water emulsion, the metabolizable lipid is vitamin E, squalene or soybean oil, which accounts for 3-15 v / v% based on the volume of the oil-in-water emulsion.

[0188] In some embodiments, the oil-in-water emulsion comprises 3-15 v / v%, preferably 4-10% v / v%, such as 5-7% v / v% squalene, as the metabolizable lipid.

[0189] In some embodiments, the oil-in-water emulsion comprises 0.3-3 v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v%, of squalene as the metabolizable oil.

[0190] In some embodiments, the oil-in-water emulsion comprises 3-10 mg / mL, such as 4-8 mg / mL, of DSPE-PEG, and DSPE-PEG is, for example, DSPE-PEG2000 or DSPE-PEG5000, used as the PEG lipid.

[0191] In some embodiments, the oil-in-water emulsion, wherein the oil-in-water emulsion contains 200-800 μg / mL, for example 300-500 μg / mL of COVID-19 recombinant virus antigen, influenza virus split antigen and / or influenza recombinant protein antigen, is used as an antigen.

[0192] In some embodiments, the oil-in-water emulsion comprises 50-250 μg / mL, such as 60-200 μg / mL, such as 70-150 μg / mL, such as 80-120 μg / mL of DDAB or DOTAP, used as the cationic polymer.

[0193] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 3-15v / v%, preferably 4-10%v / v%, for example, 5-7%v / v% of squalene, 3-10mg / mL, for example, 4-8mg / mL of DSPE-PEG, DSPE-PEG is, for example, DSPE-PEG2000 or DSPE-PEG5000, 200-800μg / mL, for example, 300-500μg / mL of COVID-19 recombinant virus antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 50-250μg / mL, for example, 60-200μg / mL, for example, 70-150μg / mL, for example, 80-120μg / mL of DDAB or DOTAP.

[0194] In some embodiments, for the oil-in-water emulsion, the oil-in-water emulsion includes 0.3-3 v / v%, for example, 0.4-2.5% v / v%, for example, 0.5-2% v / v% squalene, 3-10 mg / mL, for example, 4-8 mg / mL DSPE-PEG, DSPE-PEG is, for example, DSPE-PEG2000 or DSPE-PEG5000, 200-800 μg / mL, for example, 300-500 μg / mL of COVID-19 recombinant virus antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 40-500 μg / mL, 50-400 μg / mL, for example, 60-200 μg / mL, for example, 70-150 μg / mL, for example, 80-120 μg / mL of DDAB or DOTAP.

[0195] In some embodiments, the present application provides an oil-in-water emulsion, wherein the antigen is one or more influenza virus split antigens, including H1N1, H3N2 and BV influenza strains, and the antigen concentration is, for example, 300ug / ml.

[0196] In some embodiments, the present application provides an oil-in-water emulsion, wherein the antigen is one or more influenza recombinant protein antigens, for example, influenza recombinant hemagglutinin HA, including H1N1, H3N2 and BV strains, and the antigen concentration is, for example, 500ug / ml.

[0197] In some embodiments, for the oil-in-water emulsion of the present application, the antigen therein can be a single influenza strain component selected as a monovalent vaccine, or can be combined with multiple different types of influenza strains as a multivalent vaccine.

[0198] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 6 v / v% corn oil, 12 mg / mL DSPE-PEG 2000, 300 μg / mL influenza recombinant protein antigen, and 100 μg / mL DOTAP.

[0199] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 10-20 v / v%, such as 12-18 v / v%, such as 15 v / v% of vitamin E, 10-30 mg / mL, such as 15-25 mg / mL, such as 20 mg / mL of DMG-PEG 2000, 50-150 μg / mL, such as 75-125 μg / mL, such as 100 μg / mL of rotavirus antigen, 60-100 μg / mL, such as 70-90 μg / mL, such as 80 μg / mL of DDAB.

[0200] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 5-15v / v%, for example, 7-13v / v%, for example, 10v / v% soybean oil, 3-9mg / mL, for example, 5-8mg / mL, for example, 6mg / mL of DSPE-PEG 5000, 100-300μg / mL, for example, 150-250μg / mL, for example, 200μg / mL of RSV recombinant protein antigen, 100-300μg / mL, for example, 150-250μg / mL, for example, 200μg / mL of DOTMA.

[0201] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 3-15 v / v%, such as 4-10 v / v%, such as 5 v / v% of squalene, 2-9 mg / mL, such as 3-8 mg / mL, such as 4 mg / mL of DSPE-PEG 2000, 300-700 μg / mL, such as 400-600 μg / mL, such as 500 μg / mL of COVID-19 recombinant virus antigen, 80-160 μg / mL, such as 100-140 μg / mL, such as 120 μg / mL of DDAB.

[0202] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 4-12 v / v%, such as 6-10 v / v%, such as 8 v / v% of olive oil, 12-20 mg / mL, such as 14-18 mg / mL, such as 16 mg / mL of DSPE-PEG 5000, 30-70 μg / mL, such as 40-60 μg / mL, such as 50 μg / mL of influenza virus antigen, and 200-400 μg / mL, such as 250-350 μg / mL, such as 300 μg / mL of SM102.

[0203] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 6 v / v% corn oil, 20 mg / mL DSPE-PEG 2000, 30 μg / mL influenza recombinant protein antigen, and 600 μg / mL DOTAP.

[0204] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 8v / v% vitamin E, 90mg / mL DSPE-PEG 2000, 300μg / mL of COVID-19 inactivated virus antigen, 200μg / mL of COVID-19 recombinant protein antigen, and 800μg / mL of SM102.

[0205] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 10 v / v% soybean oil, 3 mg / mL DSPE-PEG 2000, 20 μg / mL RSV recombinant protein antigen, 2 mg / mL mucin, and 200 μg / mL protamine.

[0206] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 5 v / v% squalene, 4 mg / mL DSPE-PEG 2000, 500 μg / mL COVID-19 recombinant virus antigen, and 200 μg / mL DDAB.

[0207] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 12 v / v% olive oil, 10 mg / mL DSPE-PEG 2000, 60 μg / mL rotavirus antigen, 20 μg / mL liposomes, and 60 μg / mL DLin-MC3-DMA.

[0208] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 12 v / v% oleic acid, 50 mg / mL DSPE-PEG 2000, 10 μg / mL COVID-19 inactivated antigen, 10 μg / mL IgA antibody, and 200 μg / mL DOTMA.

[0209] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 16 v / v% squalene, 80 mg / mL DSPE-PEG 5000, 50 μg / mL influenza recombinant protein antigen, 4 mg / mL albumin, and 300 μg / mL SM102.

[0210] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 8 v / v% olive oil, 60 mg / mL DSPE-PEG 5000, 30 μg / mL rotavirus antigen, and 100 μg / mL DLin-MC3-DMA.

[0211] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 5 v / v% corn oil, 10 mg / mL DSPE-PEG 5000, 8 mg / mL mucin, and 100 μg / mL DDAB.

[0212] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 15 v / v% vitamin E, 20 mg / mL DSPE-PEG 5000, 40 μg / mL herpes zoster virus antigen, and 300 μg / mL DOTAP.

[0213] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 5 v / v% soybean oil, 8 mg / mL DSPE-PEG 5000, 30 μg / mL RSV recombinant protein antigen, 20 μg / mL IgA antibody, and 400 μg / mL protamine.

[0214] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 18 v / v% oleic acid, 6 mg / mL DSPE-PEG 5000, 500 μg / mL COVID-19 recombinant protein antigen, 40 μg / mL liposomes, and 600 μg / mL DOTMA.

[0215] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 8 v / v% corn oil, 8 mg / mL DSPE-PEG 1000, 100 μg / mL COVID-19 recombinant protein antigen, and 600 μg / mL SM102.

[0216] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 6 v / v% olive oil, 10 mg / mL DSPE-PEG 1000, 80 μg / mL herpes zoster protein antigen, 10 mg / mL albumin, and 400 μg / mL DOTMA.

[0217] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 4 v / v% vitamin E, 6 mg / mL DSPE-PEG 1000, 800 μg / mL of COVID-19 inactivated virus antigen, 15 μg / mL of VLP, and 200 μg / mL of DOTAP.

[0218] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 15 v / v% soybean oil, 5 mg / mL DSPE-PEG 1000, 60 μg / mL RSV recombinant protein antigen, 15 μg / mL IgA antibody, and 100 μg / mL protamine.

[0219] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 8 v / v% oleic acid, 40 mg / mL DSPE-PEG 1000, 100 μg / mL rotavirus antigen, 16 μg / mL extracellular vesicles, and 120 μg / mL DLin-MC3-DMA.

[0220] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 10 v / v% squalene, 2 mg / mL DSPE-PEG 1000, 30 μg / mL COVID-19 recombinant protein antigen, 50 μg / mL COVID-19 inactivated virus antigen, and 600 μg / mL DDAB.

[0221] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 2 v / v% corn oil, 12 mg / mL DSPE-PEG 1000, 100 μg / mL influenza recombinant protein antigen, 4 mg / mL mucin, and 100 μg / mL SM102.

[0222] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 4 v / v% olive oil, 5 mg / mL DME-PEG 1000, 20 μg / mL rotavirus antigen, 8 mg / mL albumin, and 500 μg / mL DOTMA.

[0223] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 20 v / v% vitamin E, 60 mg / mL DPPC, 120 μg / mL RSV recombinant protein antigen, and 400 μg / mL DOTAP.

[0224] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 2 v / v% soybean oil, 80 mg / mL DPPE, 60 μg / mL IgA antibody, and 300 μg / mL protamine.

[0225] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion includes 4 v / v% oleic acid, 16 mg / mL DSPE-PEG 1000, 200 μg / mL herpes zoster antigen, 60 μg / mL liposomes, and 200 μg / mL DLin-MC3-DMA.

[0226] In some embodiments, the present application provides an oil-in-water emulsion, wherein the oil-in-water emulsion comprises 20 v / v% squalene, 50 mg / mL DSPE-PEG 1000, 200 μg / mL influenza recombinant protein antigen, and 300 μg / mL DDAB.

[0227] On the other hand, the present application provides a method for preparing an oil-in-water emulsion, which comprises the following steps: configuring the lipid or lipid-like substance and the antigen, natural protein or antibody, or any combination thereof into an aqueous phase, dissolving the cationic polymer or ionizable lipid or positively charged protein in a metabolizable oil to form an oil phase, and then mixing the aqueous phase and the oil phase to prepare an oil-in-water emulsion.

[0228] On the other hand, the present application provides a method for preparing an oil-in-water emulsion, which comprises the following steps: configuring the lipid or lipid-like substance into an aqueous phase, dissolving the cationic polymer or ionizable lipid or positively charged protein in a metabolizable oil to configure it into an oil phase, and then mixing the aqueous phase and the oil phase to prepare the oil-in-water emulsion adjuvant of the present application, and then mixing the oil-in-water emulsion adjuvant and a solution containing an antigen to prepare an oil-in-water emulsion, preferably, the volume ratio of the oil-in-water emulsion adjuvant and the solution containing the antigen is 1:20-1:1, preferably 1:10-1:1 or 1:5-1:1.

[0229] On the other hand, the present application provides an immunogenic composition comprising any of the above-mentioned oil-in-water emulsion adjuvants, and an antigen, and pharmaceutically acceptable excipients as needed, wherein the immunogenic composition is used as a vaccine. Preferably, the concentration of the antigen is 10-1000 μg / mL.

[0230] In some embodiments, for the immunogenic composition, the antigen is a live attenuated virus vaccine antigen, an inactivated virus vaccine antigen, a split vaccine antigen, a virus-like particle vaccine antigen, a subunit vaccine antigen, a polysaccharide conjugate vaccine antigen, a nucleic acid vaccine antigen or a synthetic peptide vaccine antigen, for example, an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a COVID-19 recombinant virus antigen or an influenza virus antigen, an influenza virus split antigen and / or an influenza recombinant protein antigen.

[0231] On the other hand, the present application provides a method for preparing the immunogenic composition, which comprises mixing the oil-in-water emulsion adjuvant of the present invention and a solution containing an antigen, preferably, wherein the volume ratio of the oil-in-water emulsion adjuvant and the antigen-containing solution is 1:20-1:1, preferably 1:10-1:1 or 1:5-1:1.

[0232] On the other hand, the present application provides a pharmaceutical composition comprising the oil-in-water emulsion adjuvant of the present invention and a drug, and pharmaceutically acceptable excipients as needed. Preferably, the drug is, for example, 10-1000 μg / mL of an antigen, 10-10000 μg / mL of a natural protein, or 100-10000 μg / mL of an antibody, or any combination thereof.

[0233] In an eighth aspect, the present application provides the use of the oil-in-water emulsion adjuvant in the preparation of vaccines or drugs, preferably as a drug sustained-release system, drug delivery system or drug carrier.

[0234] In some embodiments, the vaccine or drug is delivered via the mucosa, preferably via the nasal mucosa.

[0235] On the other hand, the present application provides an immunogenic composition comprising the oil-in-water emulsion according to any one of the above claims and, if necessary, a pharmaceutically acceptable excipient, wherein the immunogenic composition is used as a vaccine.

[0236] On the other hand, the present application provides a pharmaceutical composition comprising the oil-in-water emulsion according to any one of the above claims, and pharmaceutically acceptable excipients as needed.

[0237] In another aspect, the present application provides a use of the oil-in-water emulsion in the preparation of a vaccine or a drug, preferably as a drug sustained-release system, a drug delivery system, or a drug carrier. In some embodiments, the vaccine or drug is delivered through the mucosa, such as the nasal mucosa or the pulmonary mucosa.

[0238] The oil-in-water emulsion adjuvant and oil-in-water emulsion provided in this application can simultaneously promote mucus penetration and prolong mucosal retention, and realize efficient and continuous transmucosal delivery of multiple antigens based on their own flexible deformation. Due to the efficient and stable transmucosal delivery, the oil-in-water emulsion adjuvant and oil-in-water emulsion have shown significantly improved mucosal immunity and systemic immune responses in various models such as the new coronavirus and influenza virus, and have excellent protection against various influenza viruses. They can protect the lung tissue of mice from infection by various respiratory viruses, demonstrating their application potential in vaccines for different mucosal infectious viruses.

[0239] Example

[0240] Example 1: Preparation of oil-in-water emulsion

[0241] The reagents and consumables used in the examples are shown in Table 1, and the instruments used are shown in Table 2. Unless otherwise specified, other reagents and consumables not mentioned were purchased from conventional reagent manufacturers in the field.

[0242] Table 1

[0243] Table 2

[0244] The experimental methods and detection methods used in the examples are as follows:

[0245] (1) Preparation of emulsion

[0246] According to the different component formulas, the emulsions were prepared by ultrasonic method, with 120W, 4 seconds on and 4 seconds off.

[0247] (2) Determination of particle size and potential distribution of emulsion

[0248] The particle size and potential distribution of the emulsion were measured by dynamic light scattering using a Zeta potential and a particle size analyzer. The specific measurement steps were as follows: the prepared emulsion was diluted 50 to 100 times, 1 to 2 mL of the diluted emulsion was added to a sample cell, and the sample was placed in a Zeta potential analyzer and a particle size analyzer (Nano Zeta Sizer, Malvern) for measurement.

[0249] (3) Determination of the assembly and entrapment efficiency of co-sonicated or adsorbed antigens or drugs in emulsions

[0250] The co-ultrasonicated emulsion or the emulsion adjuvant and antigen mixed for 30 minutes is centrifuged to remove the supernatant (select appropriate centrifugation conditions according to the size and density of the emulsion), and the concentration of the antigen or drug in the supernatant is measured, thereby indirectly calculating the amount of antigen or drug adsorbed to the emulsion surface;

[0251] The antigen or drug content is determined using a BCA or micro-BCA kit or other appropriate detection method; the antigen or drug adsorption rate is calculated according to the following formula:

[0252] Adsorption rate = (antigen or drug concentration before adsorption - antigen or drug concentration in the supernatant after adsorption) / antigen or drug concentration before adsorption × 100%.

[0253] (4) Determination of mucus stability

[0254] The emulsion prepared by co-ultrasound or the emulsion prepared by mixing the emulsion adjuvant and the antigen for 30 minutes was mixed with the mucus buffer in a ratio of 1:1, and the state of the emulsion was observed.

[0255] (5) Determination of emulsion deformability

[0256] Caco-2 cells were cultured on Transwell plates, and an emulsion containing a fluorescent antigen was added. The fluorescence intensity of the antigen in the culture medium in the lower chamber was measured to calculate the deformation efficiency of the emulsion.

[0257] (6) Detection of Young's modulus of emulsion

[0258] The prepared emulsion was dropped onto a positively charged glass slide. After adsorption, the residual emulsion was washed away, and a drop of ultrapure water was added to the sample. The Young's modulus of each group of emulsions was measured using an atomic force microscope (AFM).

[0259] (7) Animal experimental determination

[0260] Balb / c mice were provided by Vital River Biotechnology. The immunization steps involved are as follows:

[0261] a. First, mice were randomly divided into groups, with each group consisting of at least 6 mice. The mice were grouped and immunized according to the specific instructions in the examples. Immunofluorescence imaging of the mouse noses was performed at different time periods to observe the amount of antigen retained in the nasal cavity at different time periods after immunization.

[0262] b. Mice were randomly divided into groups, with at least six mice per group. Mice were grouped and immunized according to the specific instructions in the Examples. Serum and nasal and lung lavage fluid were collected and centrifuged at 10,000 g for 10 minutes. Enzyme-linked immunosorbent assay (ELISA) was used to measure IgG in serum and IgA in lavage fluid, respectively, to assess mucosal and systemic immune responses.

[0263] c. Mice were randomly divided into groups, with each group consisting of six or more mice. Mice were grouped and immunized according to the specific instructions in the Examples. After the first vaccination, the mice were challenged with influenza virus. Three days after the challenge, lung tissues were collected, ground and centrifuged, and the viral load in the tissues was measured using real-time quantitative polymerase chain reaction (RT-QPCR).

[0264] The components used in the examples are shown in Table 3.

[0265] Table 3

[0266] Emulsions were prepared according to the different components in Table 3. Components a and b were dissolved in water and uniformly dispersed by ultrasonication for 1 min to obtain an aqueous suspension containing components a and b. Component c was dissolved in the oil phase.

[0267] Use a pipette to draw up the oil phase (D) containing dissolved component C at the corresponding volume ratio shown in Table 3. For example, 5% squalene means 50 μl of squalene is required per 1 mL of the system. This is added to the aqueous suspension to a total volume of 1 mL. Ultrasonic emulsification (120 W, 2 minutes, 4 seconds on, 4 seconds off) is then used to prepare an oil-in-water emulsion. The final emulsion contains approximately 1-100 mg / mL of component A and approximately 1-1000 μg / mL of component C.

[0268] Example 2: Assembly and Encapsulation Efficiency of Antigens in Emulsions of Different Schemes

[0269] Emulsions were prepared according to the different components in Table 3. The horizontal axis is the corresponding solution number in Table 3. Component a was dissolved in water, and component c was dissolved in the oil phase. Ultrasonic dispersion was performed for 1 minute to uniformly disperse the emulsion. The total volume of the emulsion system was 1 mL. Ultrasonic emulsification (120W, 2 minutes, 4 seconds on, 4 seconds off) was used to prepare the oil-in-water emulsion. Antigen loading efficiency was detected using detection method 3. The results in Figure 1 show that the antigen loading efficiency was consistent across the different formulations, all greater than 95%.

[0270] Example 3: Mucus Stability

[0271] The mucus stability test mainly verifies whether the emulsion can exist stably in the mucus. If it is stable, it is possible to penetrate the mucus. If it is unstable and breaks the emulsion, it is no different from a simple antigen and the next step of delivery cannot be achieved.

[0272] The mucus stability results are shown in Figure 2. When only component C was present, the emulsion demulsified when mixed with mucus (Table 3, Figure 3, Sample 26), indicating that when only component C was present in the system, the system was unstable in the mucus system and could not be further delivered. When only component A was present, sample 27 was stable in the mucus system, but the emulsion itself was unstable and easily stratified. Moreover, in the mucosal residence time shown in Figure 3, samples 26 and 27 showed almost no significant increase compared to the antigen alone. In addition, in the transmembrane efficiency shown in Figure 5, samples 26 and 27 showed significantly lower efficiency than sample 3. However, when a solution containing components A and C was used (Table 3, Figure 3, Sample 4), the system remained stable when mixed with mucus, indicating that components A and C together stabilize the emulsion in mucus for further delivery. This example demonstrates that components A and C are necessary to maintain the stability of the emulsion in mucus and prolong its mucosal residence time. The mucus stability tests of other formulations are shown in Table 4 . It can be seen that the formulations in Table 3 containing both PEG lipids and cationic lipids can achieve better mucus stability.

[0273] The scoring criteria are as follows:

[0274] Demulsification: 1 point (e.g., Figure 3, sample No. 26)

[0275] The emulsion is unstable and easily separates: 3 points (e.g., Figure 3, sample No. 27)

[0276] Emulsion intact: 5 points (e.g., sample No. 4 in Figure 3)

[0277] Table 4. Mucus stability test

[0278] Example 4: Mucosal residence time determination

[0279] Sufficient residence time of the emulsion in the mucosa means that the antigen can be better delivered. The emulsion was prepared according to the different components in Table 3. The groups are the corresponding scheme numbers in Table 3. Component A was dissolved in water and component C was dissolved in the oil phase. Ultrasonication was performed for 1 minute to make it uniformly dispersed. The total volume of the emulsion system was 1 mL. The water-in-oil emulsion was prepared by ultrasonic emulsification (120W, 2 minutes, ultrasonication for 4 seconds and stop for 4 seconds). The mucosal residence time of the loaded emulsion was tested using detection method 7. The results are shown in Figure 3 ， This shows that the emulsion stabilized by components A+C can significantly prolong the residence time of the antigen in the mucosa.

[0280] Example 5: Transmucosal Delivery (Variation)

[0281] Emulsions were prepared according to the different components listed in Table 3, with the components designated by the corresponding protocol numbers. Component a was dissolved in water, and component c was dissolved in the oil phase. Ultrasonic dispersion was performed for 1 minute to achieve uniform dispersion. The total volume of the emulsion was 1 mL. Ultrasonic emulsification (120W, 2 minutes, 4 seconds on, 4 seconds off) was used to prepare the oil-in-water emulsion. Fluorescence intensity and Young's modulus in the lower chamber of the Transwell were measured using assays 5 and 6.

[0282] For comparison, hard particles with a similar particle size to the emulsion were prepared using albumin as the raw material. The specific preparation method was as follows: a 20 mg / mL albumin solution was prepared using a 10 mM NaCl aqueous solution at a pH of 9.8 as a buffer. 2 mL of the albumin solution was then mixed with 2 mL of anhydrous ethanol and magnetically stirred for 10 minutes. Then, 4 mL of anhydrous ethanol was added dropwise at a rate of 2 mL / min (total ethanol:albumin = 3:1 volume ratio). 160 μL of an 8% glutaraldehyde aqueous solution was then added, cross-linked, and cured for 24 hours. The resulting sample was centrifuged at 2,000 g for 20 minutes, and the supernatant was centrifuged at 20,000 g for 20 minutes. The sample was washed twice to obtain solid albumin particles. The particle size was determined according to Test Method 2 and was approximately 300 nm, similar to the emulsion particle size. As shown in Figure 4, the emulsion exhibited a smaller Young's modulus and flexible deformation properties. In addition, the results in Figure 5 show that hard particles cannot penetrate the mucosal layer. The efficiency of samples 26 and 27 is very low. The transmucosal efficiency of other emulsion formulas is greater than 80%, indicating that the emulsion with simultaneous stabilization of components A+C can better achieve transmucosal cell delivery.

[0283] It is noteworthy that when the system lacks a cationic lipid component (i.e., Sample No. 27 in Table 3), the membrane penetration efficiency of the emulsion is significantly reduced (as shown in Figure 5). This indicates that cationic lipids are very important for the membrane penetration efficiency of the emulsion of the present invention.

[0284] Example 6: Preparation of oil-in-water emulsions under different conditions

[0285] The tight junctions between mucosal epithelial cells are about 10-20nm. The emulsion has good deformation properties due to its oily core, but its deformation capacity is about 3-6 times. Therefore, after satisfying the dual properties of mucus penetration and mucosal adhesion, it is easier to cross the cell barrier and achieve transmucosal delivery when the emulsion particle size is smaller.

[0286] According to the different component formulas in Table 3, different cationic lipids, PEG lipids, PEG molecular weight lengths, oil phases, oil phase concentrations, and different mass ratios of component C to component A were set. The above conditions were changed separately to prepare emulsions. Component a was dissolved in water, component c was dissolved in the oil phase, and the total volume of the emulsion system was 1 mL. The oil-in-water emulsion was prepared by ultrasonic emulsification (120 W, 2 min, 4 seconds on, 4 seconds off).

[0287] The different emulsions successfully prepared were diluted about 100-300 times, and the particle size of the emulsions was measured by dynamic light scattering using Zeta potential and particle size analyzer. The specific measurement steps were as follows: 1-2 mL of the diluted emulsion was added to the sample cell, and placed in a Zeta potential analyzer and a particle size analyzer (Nano Zeta Sizer, Malvern) for measurement. The results are shown in Figures 6-11. DDAB can be selected as the cationic lipid, DSPE-PEG can be selected as the PEG lipid, and PEG2000 can be selected as the PEG molecular weight; 5-10% squalene can be selected as the oil phase, and a smaller particle size can be obtained when the C:A ratio is selected to be C:A≈1:33.

[0288] Example 7: Mucosal and systemic immune effects of nasal spray immunization

[0289] Mucosal immunity is a crucial component of the body's immune defense. IgA antibodies are the primary effector molecules of mucosal immunity, primarily found in mucosal secretions, such as nasal and lung washes. IgA antibodies specifically bind to and neutralize pathogens, preventing them from further infecting the body. High levels of IgA antibodies on mucosal surfaces indicate a strong mucosal immune system, effectively combating viral invasion. In contrast, IgG antibodies are one of the primary effector molecules of systemic immunity, widely present in serum. They possess multiple functions, including antiviral, antibacterial, toxin-neutralizing, and immunomodulatory. When a virus invades the body, IgG antibodies rapidly recognize and bind to the virus, preventing it from further infecting cells. Therefore, higher serum IgG levels indicate a stronger systemic immune system, enabling a more effective response to viral invasion. In summary, mucosal and systemic immunity play distinct roles in the body's immune defense, with IgA and IgG antibodies serving as their primary effector molecules, respectively. By detecting the levels of IgA antibodies in nasal and lung washes and IgG antibodies in serum, the body's mucosal immune and systemic immune responses can be assessed.

[0290] Emulsions were prepared according to the different components listed in Table 3, with the groups being designated by the corresponding protocol numbers. Component A was dissolved in water, and component C was dissolved in the oil phase. Ultrasonic dispersion was performed for 1 minute to achieve uniform dispersion. The total volume of the emulsion system was 1 mL. Ultrasonic emulsification (120W, 2 minutes, 4 seconds on, 4 seconds off) was used to prepare the oil-in-water emulsion. During the sonication process, the COVID-19 recombinant spike protein was added. Its particle size was 300±50 nm, its potential was 0±10 mV, and its Young's modulus was 20-400 MPa. Mice were inoculated with 10 μL of the emulsion per nostril via nasal instillation or spray on days 0 and 14. Serum was collected from the mice on day 28 for IgG titer. Nasal and lung lavage fluid was also isolated for IgA titer. Antibody titers were determined by enzyme-linked immunosorbent assay. Aluminum adjuvant served as a positive control. The results are shown in Figure 12. The IgG antibodies specific to the new coronavirus S protein in the serum and the IgA antibodies specific to the new coronavirus S protein in the nasal and lung washes were significantly increased due to various formulations. In particular, the IgA antibodies in the nasal and lung washes of the formulations in this study were significantly higher than that of the positive control group aluminum adjuvant, indicating that the emulsion stabilized by components A+C can significantly enhance humoral immunity and mucosal immune responses.

[0291] Example 8: Effect of mucosal vaccines based on influenza antigens

[0292] Emulsions were prepared according to the different components in Table 3. The groups were the corresponding scheme numbers in Table 3. Component a was dissolved in water, component c was dissolved in the oil phase, and ultrasonicated for 1 minute to disperse it evenly. The total volume of the emulsion system was 1 mL. Ultrasonication (120W, 2 minutes, ultrasonication for 4 seconds and stop for 4 seconds) was used to prepare the water-in-oil emulsion. The influenza recombinant antigen was added during the ultrasonication process. The mice were inoculated into the nasal cavity on days 0 and 14 by nasal drip, with 10 microliters of emulsion in each nostril. The mouse serum was taken on day 28 to detect the IgG titer. The nasal and lung lavage fluid was separated to detect the IgA titer. The antibody titer was detected by enzyme-linked immunosorbent assay. Aluminum adjuvant was the positive control group. The results are shown in Figure 13 ， The results showed that various formulations caused significant increases in influenza HA protein-specific IgG antibodies in serum and influenza HA protein-specific IgA antibodies in nasal and lung washes. In particular, the IgA antibodies in nasal and lung washes of the formulation in this study were significantly higher than that of the positive control group aluminum adjuvant, indicating that the emulsion stabilized by components A+C can significantly enhance humoral immunity and mucosal immune responses.

[0293] Example 9: Mucosal immune effects of aerosolized pulmonary delivery

[0294] According to the different components in Table 3, the emulsion was prepared. The groups were the corresponding scheme numbers in Table 3. Component a was dissolved in water, component c was dissolved in the oil phase, and ultrasonicated for 1 minute to make it evenly dispersed. The total volume of the emulsion system was 1 mL. Ultrasonic emulsification (120W, 2 minutes, supersonic for 4 seconds and stop for 4 seconds) was used to prepare the water-in-oil emulsion. The COVID-19 recombinant antigen was added during the ultrasonication process. The mouse nasal cavity was inoculated with 10 microliters of emulsion per nostril in the form of a spray on days 0 and 14. The IgA titer of the lung lavage fluid was isolated on day 28. The antibody titer was detected by enzyme-linked immunosorbent assay. Aluminum adjuvant was used as the positive control group. The results are shown in Figure 14 ， This indicates that multiple formulations administered by spray can significantly increase antigen-specific IgA antibodies in lung lavage fluid, indicating that emulsions stabilized by components A+C can significantly enhance mucosal immune responses.

[0295] Example 10: When component B is an enterovirus antigen (rotavirus antigen), mucosal immunity of the vagina and intestinal mucosa (remote mucosal immunity) is achieved

[0296] Emulsions were prepared according to the different components in Table 3, with the groups being the corresponding solution numbers in Table 3. Component A was dissolved in water, and component C was dissolved in the oil phase. Ultrasound was applied for 1 minute to achieve uniform dispersion. The total volume of the emulsion system was 1 mL. Ultrasound (120W, 2 minutes, 4 seconds on, 4 seconds off) was used to prepare the oil-in-water emulsion. Enterovirus antigen was added during the ultrasound process. The mice were inoculated into the nasal cavity via nasal drops on days 0 and 14, with 10 microliters of emulsion per nostril. On day 28, small intestinal lavage fluid was isolated and tested for IgA titers. Antibody titers were detected using enzyme-linked immunosorbent assay (ELISA). Aluminum adjuvant served as the positive control group. The results, shown in Figure 15, indicate that various formulations significantly increased antigen-specific IgA antibodies in the distal intestinal mucosa, indicating that the emulsion stabilized by components A and C can significantly enhance the distal mucosal immune response.

[0297] Example 11: When component B is influenza virus antigen, good lung protection effect is achieved

[0298] According to the different groups in Table 3, the emulsion was prepared. The group was the corresponding scheme number in Table 3. Component a was dissolved in water, and component c was dissolved in the oil phase. Ultrasonic 1min was used to disperse it evenly. The total volume of the emulsion system was 1mL. Ultrasonic (120W, 2min, ultrasonic 4 seconds and 4 seconds) emulsification was used to prepare an oil-in-water emulsion. Influenza virus HA antigen was added during the ultrasonic process. In the form of nasal drops, the nasal cavity of mice was inoculated on day 0 and day 14, with 10 microliters of emulsion per nostril. On day 28, mice were subjected to an influenza virus challenge experiment, and RT-QPCR was used to detect the viral load in lung tissue. The listed injection vaccine quadrivalent influenza virus split vaccine (from Sinovac Biotech) was the positive control group. As shown in Figure 16, it was shown that a variety of formulas can also significantly reduce the influenza virus load in lung tissue, indicating that the emulsion stabilized by components A+C can protect lung tissue from viral infection.

[0299] Example 12: Delivery of small molecules (taking Scheme 4 in Table 3 as an example)

[0300] Hydrophilic CpG (TLR9 ​​agonist) and hydrophobic MPLA (monophosphoryl lipid A) can be added to the solution before or after emulsion formation through adsorption or entrapment, with no effect on emulsion particle size or stability. Furthermore, the addition of hydrophilic and hydrophobic small molecules has no effect on emulsion stability.

[0301] Component a was dissolved in water, and component c was dissolved in the oil phase. Ultrasonication was performed for 1 minute to achieve uniform dispersion. The total volume of the emulsion system was 1 mL. An oil-in-water emulsion was prepared using ultrasonic emulsification (120W, 2 minutes, 4 seconds on, 4 seconds off). In this experiment, component a was DSPE-PEG-2k and component c was DDAB. The COVID-19 recombinant antigen was added during the ultrasonication process. On days 0 and 14, mice were inoculated with 10 μL of emulsion per nostril via nasal instillation. On day 28, mouse serum was collected for IgG titer. Nasal and lung lavage fluid was isolated for IgA titer. Antibody titer was measured using enzyme-linked immunosorbent assay. Aluminum adjuvant served as the positive control. As shown in Figure 17, the addition of small molecule stimulants to various formulations significantly increased antigen-specific IgG antibodies in serum and IgA antibodies in nasal and lung lavage fluid, indicating that the introduction of immunostimulants to the emulsion stabilized by components A and C significantly enhanced humoral and mucosal immune responses.

[0302] Example 13: Safety Characterization (Taking Scheme 4 in Table 3 as an Example)

[0303] Component a was dissolved in water, component c was dissolved in the oil phase, and ultrasonication was performed for 1 minute to uniformly disperse the emulsion. The total volume of the emulsion system was 1 mL. Ultrasonication (120W, 2 minutes, ultrasonication for 4 seconds on and 4 seconds off) was used to prepare an oil-in-water emulsion. In this experiment, component a was DSPE-PEG-2k and component c was DDAB. COVID-19 recombinant antigen was added during the ultrasonication process. The mice were inoculated into the nasal cavity by nasal drip on days 0 and 14, with 10 microliters of emulsion per nostril. On day 28, blood was collected from the mice for testing of serum biochemical indicators. The control group was the PBS group, i.e., the negative control group, and the aluminum adjuvant was the positive control group. The results are shown in Figure 18. Compared with the PBS nasal drop group and the aluminum adjuvant injection group, the emulsion nasal drop group did not show obvious heart, liver, spleen, lung, and kidney toxicity. Similarly, the emulsion nasal drop groups of schemes 1-3 and 5-24 in Table 3 also did not show obvious heart, liver, spleen, lung, and kidney toxicity.

[0304] Example 14 Preparation of oil-in-water emulsion adjuvant

[0305] The components of the oil-in-water emulsion adjuvant are shown in Table 5.

[0306] Table 5

[0307] Emulsion adjuvants were prepared according to the different components in Table 5. Component a was dissolved in water and uniformly dispersed by ultrasonication for 1 min to obtain an aqueous suspension containing component a. Component b was dissolved in the oil phase.

[0308] Use a pipette to draw up the oil phase (D) containing dissolved component c at the corresponding volume ratio in Table 5. For example, 5% squalene means 50 μl of squalene is required per 1 mL of the system. This is added to the aqueous suspension to a total volume of 1 mL. Ultrasonic emulsification (120 W, 2 min, 4 seconds on, 4 seconds off) is then used to prepare an oil-in-water emulsion adjuvant. The final emulsion adjuvant contains approximately 2-20 mg / mL of component a and approximately 30-400 μg / mL of component b.

[0309] Example 15: Assembly and Encapsulation Efficiency of Antigens in Emulsions of Different Schemes

[0310] Prepare emulsion adjuvant according to different components in table 5, horizontal coordinate is corresponding scheme number in table 5, and a component is dissolved in water, and b component is dissolved in oil phase, and emulsion system cumulative volume is 1mL, adopts ultrasonic (120W, 2min, surpasses 4 seconds and stops 4 seconds) emulsifying method, prepares oil-in-water emulsion.The emulsion of different recipes and antigen blend, wherein altogether ultrasonic group is that antigen and emulsion are altogether ultrasonic, detects antigen loading efficiency.Figure 19 shows, and after multiple recipes and influenza virus HA protein blend 30min, is loaded in efficiency consistent (wherein altogether ultrasonic is that 4 recipes in table 3 are altogether ultrasonic and make).Result shows in different recipes, and blended antigen does not affect antigen loading efficiency.

[0311] Example 16: Mucus Stability

[0312] In Formulation No. 3 in Table 5, component A was dissolved in water, and component B was dissolved in the oil phase. Ultrasonication was applied for 1 minute to achieve uniform dispersion. The total volume of the emulsion system was 1 mL, and an oil-in-water emulsion adjuvant was prepared using ultrasonic emulsification (120W, 2 minutes, 4 seconds on, 4 seconds off). After the emulsion adjuvant was blended with the antigen, it was then blended with mucus. The emulsion remained stable in the mucus, with no demulsification or stratification, indicating that the antigen blend did not affect the mucus stability of the emulsion.

[0313] In Figure 20, sample No. 3 in Table 5 was mixed with influenza HA antigen for 30 minutes and then mixed with mucus. The emulsion was stable, indicating that the antigen blend did not affect the mucus stability of the emulsion. The mucus stability scores in Table 6 show consistent results.

[0314] The scoring criteria are as follows:

[0315] Demulsification: 1 point (e.g., Figure 3, sample No. 26)

[0316] The emulsion is unstable and easily separates: 3 points (e.g., Figure 3, sample No. 27)

[0317] Emulsion intact: 5 points (e.g., Figure 3, sample No. 3)

[0318] Table 6. Mucus stability test

[0319] Example 17: Mucosal residence time determination

[0320] Emulsion adjuvants were prepared according to the different components in Table 5. The groups were the corresponding scheme numbers in Table 5. Component A was dissolved in water, and component B was dissolved in the oil phase. Ultrasonication was performed for 1 minute to uniformly disperse the components. The total volume of the emulsion system was 1 mL. Ultrasonication (120W, 2 minutes, 4 seconds on, 4 seconds off) was used to prepare the water-in-oil emulsion adjuvant. The mucosal residence time of the loaded emulsion was tested using detection method 7. The results are shown in Figure 21. ，The emulsion adjuvant mixed with antigen did not affect the mucosal residence time of the antigen. The residence time of samples 3-5 in Table 5 after mixing with antigen was consistent with that of the co-ultrasound samples, indicating that the antigen mixing had no effect on the mucosal residence time.

[0321] Example 18: Transmucosal Delivery (Variation)

[0322] Emulsions were prepared according to the different components in Table 5. The groups were the corresponding solution numbers in Table 5. Component a was dissolved in water and component b was dissolved in the oil phase. Ultrasonication was performed for 1 minute to uniformly disperse the components. The total volume of the emulsion system was 1 mL. Ultrasonication (120W, 2 minutes, 4 seconds on and 4 seconds off) was used to prepare the water-in-oil emulsion. The fluorescence intensity in the lower chamber of the Transwell was detected using detection method 5. The results are shown in Figure 22. ， The blending of antigens into the emulsion did not affect the transmembrane efficiency of the emulsion. The transmucosal cell delivery efficiency of various formulations blended with antigens in Table 5 was consistent with that of the co-ultrasound formulation, indicating that antigen blending had no effect on the transmucosal efficiency.

[0323] Example 19: Mucosal and systemic immune effects of nasal spray immunization

[0324] According to the different components in Table 5, the emulsion adjuvant was prepared. The groups were the corresponding scheme numbers in Table 5. Component a was dissolved in water, component b was dissolved in the oil phase, and ultrasonicated for 1 minute to disperse it evenly. The total volume of the emulsion system was 1 mL. The water-in-oil emulsion adjuvant was prepared by ultrasonication (120W, 2 minutes, ultrasonication for 4 seconds and stop for 4 seconds). The emulsion adjuvant of each formula was then blended with the influenza virus split antigen for 30 minutes. The mouse nasal cavity was inoculated with 10 microliters of emulsion per nostril on days 0 and 14 by nasal drip or spray. On day 28, the mouse serum was taken to detect the IgG titer. The nasal and lung lavage fluid was separated to detect the IgA titer. The antibody titer was detected by enzyme-linked immunosorbent assay. The results are shown in Figure 23. The influenza HA protein-specific IgG antibodies in the serum and the new crown S protein-specific IgA antibodies in the nasal and lung lavage fluid were significantly increased by various formulas, which is consistent with the co-ultrasound effect and can significantly enhance the humoral and mucosal immune responses.

[0325] Example 20: Effect of mucosal vaccines based on the novel coronavirus Spike antigen

[0326] Emulsion adjuvants were prepared according to the different components in Table 5, with the groups being the corresponding scheme numbers in Table 5. Component a was dissolved in water, component b was dissolved in the oil phase, and ultrasonication was performed for 1 minute to uniformly disperse the emulsion. The total volume of the emulsion system was 1 mL, and an ultrasonic (120W, 2 minutes, ultrasonication for 4 seconds on, 4 seconds off) emulsification method was used to prepare an oil-in-water emulsion adjuvant. The emulsion adjuvants of each formula were then blended with the new coronavirus Spike protein antigen for 30 minutes. The mice were inoculated into the nasal cavity on days 0 and 14 by nasal instillation or spray, with 10 microliters of emulsion per nostril. On day 28, mouse serum was collected to test IgG titers. Nasal and lung lavage fluid was separated to test IgA titers. Antibody titers were detected using enzyme-linked immunosorbent assay. The results are shown in Figure 24. The various formulas caused a significant increase in IgG antibodies specific to the new coronavirus Spike protein in serum and IgA antibodies specific to the new coronavirus S protein in nasal and lung lavage fluids, consistent with the co-ultrasound effect, and were able to significantly enhance humoral and mucosal immune responses.

[0327] Example 21: Achieving good lung protection against influenza virus

[0328] According to the different components in Table 5, the emulsion adjuvant was prepared. The groups were the corresponding scheme numbers in Table 5. Component a was dissolved in water, component b was dissolved in the oil phase, and ultrasonic 1 min was used to disperse it evenly. The total volume of the emulsion system was 1 mL. The water-in-oil emulsion adjuvant was prepared by ultrasonic emulsification (120W, 2 min, ultrasonic 4 seconds on, 4 seconds off). Each formula emulsion adjuvant and influenza A strain split antigen were then blended for 30 min. The mice were inoculated into the nasal cavity on days 0 and 14 by nasal drops, with 10 microliters of emulsion per nostril. On day 28, the mice were subjected to an influenza virus challenge experiment, and the viral load in the lung tissue was detected by RT-QPCR. The listed injectable vaccine quadrivalent influenza virus split vaccine (from Sinovac Biotech) was the positive control group. As shown in Figure 25, multiple formulas can also significantly reduce the influenza virus load in the lung tissue, which is consistent with the co-ultrasound effect and can protect the lung tissue from viral infection.

[0329] Example 22: Achieving good lung protection against influenza virus

[0330] According to the different components in Table 5, the emulsion adjuvant was prepared. The groups were the corresponding scheme numbers in Table 5. Component a was dissolved in water, component b was dissolved in the oil phase, and ultrasonic 1 min was used to disperse it evenly. The total volume of the emulsion system was 1 mL. The water-in-oil emulsion adjuvant was prepared by ultrasonic emulsification (120W, 2 min, ultrasonic 4 seconds on, 4 seconds off). Each formula emulsion adjuvant and influenza B strain split antigen were then blended for 30 min. The mice were inoculated into the nasal cavity on days 0 and 14 by nasal drops, with 10 microliters of emulsion per nostril. On day 28, the mice were subjected to an influenza virus challenge experiment, and the viral load in the lung tissue was detected by RT-QPCR. The listed injectable vaccine quadrivalent influenza virus split vaccine (from Sinovac Biotech) was the positive control group. As shown in Figure 26, multiple formulas can also significantly reduce the influenza virus load in the lung tissue, which is consistent with the co-ultrasound effect and can protect the lung tissue from viral infection.

Claims

1. An oil-in-water emulsion, which comprises an oil phase and a water phase, The water phase comprises: 0.5 - 100 mg / mL of PEG lipid or lipid-like substance, and 10 - 1000 μg / mL of antigen, or 10 - 10000 μg / mL of natural protein or 100 - 10000 μg / mL of antibody, or any combination thereof; and The oil phase comprises: 30 - 1100 μg / mL of cationic polymer or ionizable lipid or positively charged protein, wherein the cationic polymer is preferably a cationic lipid, and the pKa of the ionizable lipid or positively charged protein is less than 6.8; and Metabolizable oil, preferably 0.3 - 25 v / v% based on the volume of the oil-in-water emulsion; Wherein, the Young's modulus of the oil-in-water emulsion is 20 - 800 MPa; the average particle size of the emulsion droplets in the oil-in-water emulsion is 50 - 800 nm.

2. The oil-in-water emulsion according to claim 1, wherein the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diacylglycerol-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dodecyloxy-polyethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposome, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), liposome, LNP and extracellular exosome. Preferably, the molecular weight of polyethylene glycol in the PEG lipid is 550 - 8000, preferably, the molecular weight of polyethylene glycol in the PEG lipid is 1000 - 5000, for example, 1500 - 2500.

3. The oil-in-water emulsion according to claim 1, wherein the antibody or antigen or natural protein is selected from one or more of the following: live attenuated virus vaccine antigens such as influenza virus, COVID-19 virus, hand-foot-and-mouth virus, rotavirus, inactivated virus vaccine antigens, split vaccine antigens, virus recombinant protein antigens, human serum albumin, genetically recombinant or purified antibodies IgG, IgM, IgA, nanobody, diabody, pharmaceutical protein.

4. The oil-in-water emulsion according to claim 1, wherein the cationic polymer or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecylammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine polyoxyethylene ether bisquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-bis(octadecyloxy)-3-methylammonium propane (chloride, DOTMA), 4-(N,N-dimethylamino)butyric acid (dilinoleoyl) methyl ester (DLin-MC3-DMA), octadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM-102), protamine or cationic polypeptide, and PEI.

5. The oil-in-water emulsion according to claim 1, wherein the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, dimethyl silicone oil, isopropyl laurate, and glyceryl tricaprate.

6. The oil-in-water emulsion according to claim 1, wherein the PEG lipid is DSPE-PEG or DMG-PEG, such as DSPE-PEG 5000, DSPE-PEG 2000 or DMG-PEG-2000, wherein, The cationic polymer is a cationic lipid, such as DDAB, DOTAP or DC-CHOL, wherein the antigen is, for example, an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a varicella-zoster antigen, a COVID-19 recombinant virus antigen or an influenza virus antigen, wherein the metabolizable oil is vitamin E, squalene or soybean oil, which is 3-15 v / v% based on the volume of the oil-in-water emulsion, or 0.3-3 v / v% based on the volume of the oil-in-water emulsion, for example 0.4-2.5% v / v%, for example 0.5-2% v / v%.

7. The oil-in-water emulsion according to claim 1, wherein the oil-in-water emulsion comprises, based on the volume of the oil-in-water emulsion, 3-15 v / v%, preferably 4-10% v / v%, for example 5-7% v / v% of squalene or soybean oil, 3-10 mg / mL, for example 4-8 mg / mL of DSPE-PEG, DSPE-PEG being, for example, DSPE-PEG2000 or DSPE-PEG5000, 200-800 μg / mL, for example 300-500 μg / mL of COVID-19 recombinant viral antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 30-1000 μg / mL, for example 50-900 μg / mL, for example, 30-450 μg / mL, for example 600-900 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL of DDAB or DOTAP; or, wherein the oil-in-water emulsion comprises, based on the volume of the oil-in-water emulsion, 0.3-3 v / v%, for example 0.4-2.5% v / v%, for example 0.5-2% v / v% of squalene or soybean oil, 3-10 mg / mL, for example 4-8 mg / mL of DSPE-PEG, DSPE-PEG being, for example, DSPE-PEG2000 or DSPE-PEG5000, 200-800 μg / mL, for example 300-500 μg / mL of COVID-19 recombinant viral antigen, influenza virus split antigen and / or influenza recombinant protein antigen, 30-1000 μg / mL, for example 50-900 μg / mL, for example, 30-450 μg / mL, for example 600-900 μg / mL, for example 60-200 μg / mL, for example 70-150 μg / mL, for example 80-120 μg / mL of DDAB or DOTAP.

8. An immunogenic composition or a pharmaceutical composition, which comprises the oil-in-water emulsion according to any one of the above claims, and pharmaceutically acceptable excipients when needed. Preferably, the immunogenic composition is used as a vaccine.

9. Use of the oil-in-water emulsion according to any one of claims 1-7 in the preparation of a vaccine or a drug, preferably as a drug sustained release system, a drug delivery system or a drug carrier. Preferably, the vaccine or the drug is delivered through the mucosa, preferably by nasal mucosa administration.

10. An oil-in-water emulsion adjuvant, which comprises an oil phase and a water phase, The water phase comprises: PEG lipid or lipid-like substance, preferably at a concentration of 0.5-50 mg / mL, preferably 0.5-20 mg / mL; The oil phase comprises: a) Cationic lipid or ionizable lipid or positively charged protein, preferably at a concentration of 30-1000 μg / mL; b) Metabolizable oil, preferably at 0.3-20 v / v% based on the volume of the oil-in-water emulsion; Among them, the Young's modulus of the oil-in-water emulsion adjuvant is 5-350 MPa, preferably 10-100 MPa. Preferably, the average particle size of the oil droplets in the oil-in-water emulsion adjuvant is 60-400 nm.

11. The oil-in-water emulsion adjuvant according to claim 10, wherein the PEG lipid or lipid-like substance is selected from one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), diacylglycerol-PEG (DAG-PEG), dioleoyl-polyethylene glycol (DAA-PEG), dimyristoyl-polyethylene glycol (DMG-PEG), octyl-polyethylene glycol (C8-PEG), dodecyloxy-polyethylene glycol (DOG-PEG), ceramide-polyethylene glycol (PEG-CER), DSPE-PEG-liposome, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), liposome, LNP and extracellular exosome. Preferably, the molecular weight of polyethylene glycol in the PEG lipid is 550-8000. Preferably, the molecular weight of polyethylene glycol in the PEG lipid is 1000-5000, such as 1500-2500.

12. The oil-in-water emulsion adjuvant according to claim 10, wherein the cationic lipid or ionizable lipid or positively charged protein is selected from one or more of the following: didodecyldimethylammonium bromide (DDAB), dodecylammonium chloride, cetyltrimethylammonium bromide, cetylpyridinium bromide, cationic polyacrylamide (CPAM), 1,2-dioleoyl-3-dimethylamino-propane (DODMA), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL), octadecylamine polyoxyethylene ether bisquaternary ammonium salt, (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), 1,2-bis(octadecyloxy)-3-methylammonium propane (chloride, DOTMA), DLin-MC3-DMA, SM-102, protamine or cationic polypeptide.

13. The oil-in-water emulsion adjuvant according to claim 10, wherein the metabolizable oil is selected from one or more of the following: corn oil, squalene, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, dimethyl silicone oil, isopropyl laurate and glyceryl tributyrate.

14. The oil-in-water emulsion adjuvant according to claim 10, wherein the PEG lipid is DSPE-PEG or DMG-PEG, such as DSPE-PEG 5000, DSPE-PEG 2000 or DMG-PEG-2000, wherein, The cationic polymer is a cationic lipid, such as DDAB, DOTAP or DC-CHOL. Among them, the metabolizable oil is vitamin E, squalene or soybean oil, which is 3-15 v / v% based on the volume of the oil-in-water emulsion adjuvant, or, alternatively, 0.3-3 v / v% based on the volume of the oil-in-water emulsion adjuvant, such as 0.4-2.5% v / v%, such as 0.5-2% v / v%.

15. The oil-in-water emulsion adjuvant according to claim 10, which comprises 0.5-20 v / v% of squalene or soybean oil, 0.5-20 mg / mL, DSPE-PEG such as DSPE-PEG2000 or DSPE-PEG5000, 30-1000 μg / mL of DDAB or DOTAP.

16. The oil-in-water emulsion adjuvant according to claim 10, which comprises 3-15 v / v%, preferably 4-10% v / v%, such as 5-7% v / v% of squalene or soybean oil, 3-10 mg / mL, such as 4-8 mg / mL of DSPE-PEG, DSPE-PEG such as DSPE-PEG2000 or DSPE-PEG5000, 40-500 μg / mL, 30-1000 μg / mL, such as 50-900 μg / mL, such as, 30-450 μg / mL, such as 600-900 μg / mL, such as 60-200 μg / mL, such as 70-150 μg / mL, such as 80-120 μg / mL of DDAB or DOTAP; or, it comprises 0.3-3 v / v%, such as 0.4-2.5% v / v%, such as 0.5-2% v / v% of squalene or soybean oil, 3-10 mg / mL, such as 4-8 mg / mL of DSPE-PEG, DSPE-PEG such as DSPE-PEG2000 or DSPE-PEG5000, 30-1000 μg / mL, such as 50-900 μg / mL, such as, 30-450 μg / mL, such as 600-900 μg / mL, such as 60-200 μg / mL, such as 70-150 μg / mL, such as 80-120 μg / mL of DDAB or DOTAP.

17. The oil-in-water emulsion adjuvant according to claim 10, wherein, The mass-volume ratio of the cationic lipid to squalene or soybean oil is 1-25 μg / μL. Preferably, the mass-volume ratio of the PEG lipid to squalene or soybean oil is 5-450 μg / μL. Preferably, the oil-in-water emulsion adjuvant comprises 0.5-15 v / v% of squalene or soybean oil, 0.5-12 mg / mL of DSPE-PEG, DSPE-PEG such as DSPE-PEG2000 or DSPE-PEG5000, 30-450 μg / mL of DDAB or DOTAP.

18. An immunogenic composition comprising an oil-in-water emulsion adjuvant according to any one of claims 10-17, an antigen, and, if necessary, a pharmaceutically acceptable excipient, wherein the immunogenic composition is used as a vaccine. Preferably, the concentration of the antigen is 10-1000 μg / mL. Preferably, the antigen is an attenuated live virus vaccine antigen, an inactivated virus vaccine antigen, a split vaccine antigen, a virus-like particle vaccine antigen, a subunit vaccine antigen, a polysaccharide conjugate vaccine antigen, a nucleic acid vaccine antigen, or a synthetic peptide vaccine antigen. For example, it is an influenza recombinant protein antigen, a rotavirus antigen, an RSV recombinant protein antigen, a herpes zoster antigen, a COVID-19 recombinant virus antigen, or an influenza virus antigen, an influenza virus split antigen, and / or an influenza recombinant protein antigen.

19. A method for preparing the immunogenic composition according to claim 18, comprising mixing the oil-in-water emulsion adjuvant according to any one of claims 10-17 and a solution containing an antigen. Preferably, the volume ratio of the oil-in-water emulsion adjuvant to the solution containing the antigen is 1:20-1:1, preferably 1:10-1:1 or 1:5-1:

1.

20. A pharmaceutical composition comprising an oil-in-water emulsion adjuvant according to any one of claims 10-17 and a drug, and, if necessary, a pharmaceutically acceptable excipient. Preferably, the drug is, for example, an antigen at 10-1000 μg / mL, a natural protein at 10-10000 μg / mL, or an antibody at 100-10000 μg / mL, or any combination thereof.

21. Use of the oil-in-water emulsion adjuvant according to any one of claims 10-17 in the preparation of a vaccine or a drug, preferably as a drug sustained-release system, a drug delivery system, or a drug carrier. Preferably, the vaccine or the drug is delivered mucosally, preferably by nasal mucosal administration.

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