Preparation method and application of emulsion type mucosal vaccine preparation

Abstract

The invention belongs to the field of vaccine development, and particularly relates to a preparation method and application of an emulsion type mucosal vaccine preparation, and the preparation method comprises the following steps: dropwise adding choline hydroxide or choline bicarbonate into an acidic compound aqueous solution, and reacting to obtain a functional aqueous phase; mixing the water phase with the oil phase and a surfactant, and carrying out ultrasonic emulsification to obtain an oil-in-water emulsion; and adding an antigen solution into the emulsion, and vortex to prepare the mucosal vaccine preparation. The mucosal vaccine is simple in preparation process, has better storage stability, is suitable for various novel mucosal immune ways such as nasal dripping and lungs, can improve the bioavailability of antigens at mucous membranes, and effectively activates mucosal immune protection of organisms.

Description

Technical Field

[0001] This invention belongs to the field of vaccine formulation research, specifically relating to a method for preparing and applying an emulsion-type mucosal vaccine formulation. Background Technology

[0002] In recent decades, various respiratory infectious diseases (such as COVID-19, influenza, and respiratory syncytial virus) have frequently occurred, exhibiting characteristics such as high infectivity and rapid mutation, seriously threatening national health. However, traditional vaccines, administered subcutaneously or intramuscularly, primarily produce IgG antibodies, which are difficult to effectively induce the production of secretory IgA (sIgA) in sites such as the nasal mucosa, thus failing to effectively prevent the replication and spread of pathogens in the host's upper respiratory tract. Nasal mucosal immunity can simultaneously stimulate local and systemic immunity at the mucosal site, inducing the production of secretory IgA (sIgA) at key infection sites such as the respiratory tract, thereby blocking the binding of pathogens to epithelial cell receptors and playing a protective role in the early stages of viral infection. The mucosal immune response can also induce the production of local tissue-resident memory (TRM) T cells to achieve long-term protection. At the same time, immune cells activated at the mucosal site can induce systemic humoral and cellular immune responses through humoral circulation, achieving multiple layers of protection. Developing novel mucosal vaccines that can effectively induce mucosal and systemic immunity and can be rapidly deployed has significant application value.

[0003] However, the nasal mucosa has complex physiological structures such as cilia with antigen clearance function, various proteases that can degrade antigens in nasal mucus, and mucosal barriers, which seriously hinder the delivery of antigens through the nasal mucosa and significantly reduce bioavailability. Effective delivery of antigens has become a huge challenge for the development of nasal vaccines.

[0004] To overcome the multiple barriers at the mucosal site, efficient delivery systems or immunostimulants have become a key strategy in the development of novel mucosal vaccines. Among these, emulsions, as a simple delivery system with some antigen universality, have become a research hotspot. Emulsions are dispersion systems composed of an oil phase, an aqueous phase, and an emulsifier. Several emulsions have already been approved as vaccine adjuvants, such as the Fluad influenza vaccine with added MF59 adjuvant. TM Humenza, a flu vaccine with added AF03 adjuvant TM However, most of the aforementioned emulsions are intramuscular injection adjuvants and have not been used in the development of mucosal vaccines. Currently, only a few emulsions are used in mucosal vaccine delivery studies and have shown good results.

[0005] CN109718372B discloses a nanoscale emulsion immunoloader PELC for nasal mucosa, which consists of the following components: an aqueous phase, an oil phase, and a sorbitan-poly(lactide-ε-caprolactone) copolymer (emulsifier). The PELC emulsion has a particle size of 20-200 nm, does not contain ionic emulsifiers, and can be used to prepare vaccine formulations that induce a strong immune response.

[0006] US12076397(B2) discloses an emulsion adjuvant W for intramuscular, intradermal, and subcutaneous administration. 80 5EC, composed of hexadecylpyridine ammonium chloride, Tween 80, ethanol, and soybean oil. Based on this, US201917292714 discloses a nanoemulsion composition with enhanced permeability, applicable to various mucosal sites such as the nose and vagina. Further development led to the NanoVax nasal mucosal delivery platform, which is used in the development of novel mucosal vaccines against influenza, viruses, and bacteria. Related patents include US202318133238, AU2023254763, WO2023US18130, US2023010121, US202117503575, EP20210771166, and US202017013024. Currently, the NanoVax nasal mucosal delivery platform has completed three Phase I clinical trials, demonstrating good biocompatibility.

[0007] The aforementioned applications demonstrate the significant potential of emulsions as mucosal vaccine delivery systems. Based on existing research, this invention synthesizes a functional aqueous phase with adhesion-promoting or penetration-promoting properties to prepare an emulsion-type mucosal vaccine formulation. Animal experiments have demonstrated that this vaccine formulation is suitable for various novel immunization methods, including nasal drops and pulmonary administration, and can significantly stimulate the body to produce mucosal immunity, cellular immunity, and humoral immunity, effectively preventing respiratory pathogens and possessing significant application value for the prevention and control of respiratory infectious diseases. Summary of the Invention

[0008] The frequent occurrence of respiratory infectious diseases in recent years has created an urgent need for mucosal vaccines. However, the multiple physiological barriers in the respiratory tract and other mucosal sites pose significant challenges to the delivery efficiency and effectiveness of mucosal vaccines. To address these problems, this invention aims to provide a method for preparing an emulsion-type mucosal vaccine formulation and its application effects in mucosal immunization.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing an emulsion-type mucosal vaccine formulation that can significantly improve antigen bioavailability, comprising:

[0011] (1) A certain amount of choline hydroxide or choline bicarbonate is added dropwise to an aqueous solution of an acidic compound, and the mixture is stirred at room temperature for 24 hours to prepare a functional aqueous phase.

[0012] (2) Weigh out the prescribed amounts of functional aqueous phase, oil phase, and surfactant according to the proportions;

[0013] (3) The functional aqueous phase, oil phase and surfactant are mixed and emulsified under ultrasonic conditions, and then sterilized by a 0.22μm filter to prepare an emulsion;

[0014] (4) Mix the emulsion and the antigen solution in a certain proportion and vortex to obtain an emulsion-type mucosal vaccine preparation.

[0015] Furthermore, the acidic compound includes any one of glycolic acid, lactic acid, nicotinic acid, geranilic acid, and glycine; preferably, the acidic compound includes any one of glycolic acid, lactic acid, and nicotinic acid.

[0016] Furthermore, the molar ratio of the choline hydroxide or choline bicarbonate to the acidic compound is 1:0.5-1:2.

[0017] Furthermore, the oil phase includes any one or a combination of at least two of squalene, soybean oil, isopropyl myristate, or tocopherol; preferably, the oil phase is any one of squalene and soybean oil.

[0018] Furthermore, the surfactant includes any one or a combination of at least two of the following: nonionic surfactants alkyl glucoside, fatty acid esters, Span, Tween, and ionic surfactants stearic acid, sulfated castor oil, and oleic acid; preferably, the surfactant is any one of Tween 80 and Tween 20.

[0019] Furthermore, the functional aqueous phase has a molar concentration of 0.5-5M, an addition amount of 50%-70% (w / w), an oil phase addition amount of 5%-45% (w / w), and a surfactant addition amount of 5%-25% (w / w); preferably, the functional aqueous phase has a molar concentration of 1M, an addition amount of 50%-60% (w / w), an oil phase addition amount of 20%-40% (w / w), and a surfactant addition amount of 5%-15% (w / w).

[0020] Furthermore, the ultrasonic power is 100-500w, and the emulsification time is 30s-5min (2 seconds for each ultrasonic operation, 3 seconds for a pause); preferably, the ultrasonic power is 200-300w, and the emulsification time is 30s-2min (2 seconds for each ultrasonic operation, 3 seconds for a pause).

[0021] Furthermore, the antigens include any one or a combination of at least two of the following disease-related antigens transmitted via the respiratory tract: coronavirus vaccine antigen, novel coronavirus vaccine antigen, influenza virus vaccine antigen, parainfluenza virus vaccine antigen, respiratory syncytial virus vaccine antigen, human metapneumovirus vaccine antigen, adenovirus vaccine antigen, measles virus vaccine antigen, herpes zoster virus vaccine antigen, pneumococcal vaccine antigen, Bordetella pertussis vaccine antigen, Mycobacterium tuberculosis, foot-and-mouth disease vaccine antigen, and Mycoplasma pneumoniae vaccine antigen.

[0022] Furthermore, the type of antigen includes any one or a combination of at least two of the following: live attenuated virus vaccine antigen, inactivated virus vaccine antigen, split vaccine antigen, virus-like particle vaccine antigen, subunit vaccine antigen, polysaccharide conjugate vaccine antigen, nucleic acid vaccine antigen, or synthetic peptide vaccine antigen.

[0023] Furthermore, the antigen solution is any one of phosphate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, and disodium hydrogen phosphate-citrate buffer; preferably, the antigen solution is any one of phosphate buffer and phosphate buffer.

[0024] Furthermore, the antigen concentration in the antigen solution is 2-1000 μg / mL.

[0025] Furthermore, the volume ratio of the emulsion to the antigen solution is 1:1 to 1:16.

[0026] Secondly, the present invention provides the application of the above-mentioned emulsion-type mucosal vaccine formulation in the development of novel mucosal vaccines.

[0027] Thirdly, the present invention provides a suitable administration method for the above-mentioned emulsion-type mucosal vaccine preparation. During use, the administration method of the emulsion-type mucosal vaccine preparation includes any one of nasal drops, pulmonary administration, nebulized inhalation, and intramuscular injection; preferably, the administration method of the vaccine preparation includes any one of nasal drops, pulmonary administration, and nebulized inhalation.

[0028] Fourthly, the present invention provides an emulsion-type mucosal vaccine formulation for nasal administration, comprising: a solution of an emulsion and an antigen in a volume ratio of 1:1 to 1:6, wherein the emulsion is prepared by:

[0029] (1) A certain amount of choline hydroxide or choline bicarbonate solution was added dropwise to an aqueous solution of an acidic compound, and the mixture was stirred at room temperature for 24 hours to prepare a functional aqueous phase.

[0030] (2) Weigh out the prescribed amounts of functional aqueous phase, oil phase, and surfactant according to the proportions;

[0031] (3) The functional aqueous phase, oil phase and surfactant are mixed and emulsified under ultrasonic conditions, and then sterilized by a 0.22μm filter to prepare an emulsion.

[0032] Furthermore, the acidic compound includes any one of glycolic acid, lactic acid, nicotinic acid, geranilic acid, and glycine; preferably, the acidic compound includes any one of glycolic acid, lactic acid, and nicotinic acid.

[0033] Furthermore, the molar ratio of the choline hydroxide or choline bicarbonate solution to the acidic compound is 1:0.5-1:2.

[0034] Furthermore, the oil phase includes any one or a combination of at least two of squalene, soybean oil, isopropyl myristate, or tocopherol; preferably, the oil phase is any one of squalene and soybean oil.

[0035] Furthermore, the surfactant includes any one or a combination of at least two of the following: nonionic surfactants alkyl glucoside, fatty acid esters, Span, Tween, and ionic surfactants stearic acid, sulfated castor oil, and oleic acid; preferably, the surfactant is any one of Tween 80 and Tween 20.

[0036] Furthermore, the functional aqueous phase has a molar concentration of 0.5-5M, an addition amount of 50%-70% (w / w), an oil phase addition amount of 5%-45% (w / w), and a surfactant addition amount of 5%-25% (w / w); preferably, the functional aqueous phase has a molar concentration of 1M, an addition amount of 50%-60% (w / w), an oil phase addition amount of 20%-40% (w / w), and a surfactant addition amount of 5%-15% (w / w).

[0037] Furthermore, the ultrasonic power is 100-500w, and the emulsification time is 30s-5min (2 seconds for each ultrasonic operation, 3 seconds for a pause); preferably, the ultrasonic power is 200-300w, and the emulsification time is 30s-2min (2 seconds for each ultrasonic operation, 3 seconds for a pause).

[0038] Furthermore, the antigens include any one or a combination of at least two of the following disease-related antigens transmitted via the respiratory tract: coronavirus vaccine antigen, novel coronavirus vaccine antigen, influenza virus vaccine antigen, parainfluenza virus vaccine antigen, respiratory syncytial virus vaccine antigen, human metapneumovirus vaccine antigen, adenovirus vaccine antigen, measles virus vaccine antigen, herpes zoster virus vaccine antigen, pneumococcal vaccine antigen, Bordetella pertussis vaccine antigen, Mycobacterium tuberculosis, foot-and-mouth disease vaccine antigen, and Mycoplasma pneumoniae vaccine antigen.

[0039] Furthermore, the type of antigen includes any one or a combination of at least two of the following: live attenuated virus vaccine antigen, inactivated virus vaccine antigen, split vaccine antigen, virus-like particle vaccine antigen, subunit vaccine antigen, polysaccharide conjugate vaccine antigen, nucleic acid vaccine antigen, or synthetic peptide vaccine antigen.

[0040] Furthermore, the antigen solution is any one of phosphate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, and disodium hydrogen phosphate-citrate buffer; preferably, the antigen solution is any one of phosphate buffer and phosphate buffer.

[0041] Furthermore, the antigen concentration in the antigen solution is 2-1000 μg / mL.

[0042] Fifthly, the present invention provides an emulsion-type mucosal vaccine formulation for pulmonary administration or nebulized inhalation, comprising: a solution of an emulsion and an antigen in a volume ratio of 1:8 to 1:16, wherein the emulsion is prepared by:

[0043] (1) A certain amount of choline hydroxide or choline bicarbonate solution was added dropwise to an aqueous solution of an acidic compound, and the mixture was stirred at room temperature for 24 hours to prepare a functional aqueous phase.

[0044] (2) Weigh out the prescribed amounts of functional aqueous phase, oil phase, and surfactant according to the proportions;

[0045] (3) The functional aqueous phase, oil phase and surfactant are mixed and emulsified under ultrasonic conditions, and then sterilized by a 0.22μm filter to prepare an emulsion.

[0046] Furthermore, the acidic compound includes any one of glycolic acid, lactic acid, nicotinic acid, geranilic acid, and glycine; preferably, the acidic compound includes any one of glycolic acid, lactic acid, and nicotinic acid.

[0047] Furthermore, the molar ratio of the choline hydroxide or choline bicarbonate solution to the acidic compound is 1:0.5-1:2.

[0048] Furthermore, the oil phase includes any one or a combination of at least two of squalene, soybean oil, isopropyl myristate, or tocopherol; preferably, the oil phase is any one of squalene and soybean oil.

[0049] Furthermore, the surfactant includes any one or a combination of at least two of the following: nonionic surfactants alkyl glucoside, fatty acid esters, Span, Tween, and ionic surfactants stearic acid, sulfated castor oil, and oleic acid; preferably, the surfactant is any one of Tween 80 and Tween 20.

[0050] Furthermore, the functional aqueous phase has a molar concentration of 0.5-5M, an addition amount of 50%-70% (w / w), an oil phase addition amount of 5%-45% (w / w), and a surfactant addition amount of 5%-25% (w / w); preferably, the functional aqueous phase has a molar concentration of 1M, an addition amount of 50%-60% (w / w), an oil phase addition amount of 20%-40% (w / w), and a surfactant addition amount of 5%-15% (w / w).

[0051] Furthermore, the ultrasonic power is 100-500w, and the emulsification time is 30s-5min (2 seconds for each ultrasonic operation, 3 seconds for a pause); preferably, the ultrasonic power is 200-300w, and the emulsification time is 30s-2min (2 seconds for each ultrasonic operation, 3 seconds for a pause).

[0052] Furthermore, the antigens include any one or a combination of at least two of the following disease-related antigens transmitted via the respiratory tract: coronavirus vaccine antigen, novel coronavirus vaccine antigen, influenza virus vaccine antigen, parainfluenza virus vaccine antigen, respiratory syncytial virus vaccine antigen, human metapneumovirus vaccine antigen, adenovirus vaccine antigen, measles virus vaccine antigen, herpes zoster virus vaccine antigen, pneumococcal vaccine antigen, Bordetella pertussis vaccine antigen, Mycobacterium tuberculosis, foot-and-mouth disease vaccine antigen, and Mycoplasma pneumoniae vaccine antigen.

[0053] Furthermore, the type of antigen includes any one or a combination of at least two of the following: live attenuated virus vaccine antigen, inactivated virus vaccine antigen, split vaccine antigen, virus-like particle vaccine antigen, subunit vaccine antigen, polysaccharide conjugate vaccine antigen, nucleic acid vaccine antigen, or synthetic peptide vaccine antigen.

[0054] Furthermore, the antigen solution is any one of phosphate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer, and disodium hydrogen phosphate-citrate buffer; preferably, the antigen solution is any one of phosphate buffer and phosphate buffer.

[0055] Furthermore, the antigen concentration in the antigen solution is 2-1000 μg / mL.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The emulsion-type mucosal vaccine preparation provided by the present invention has fewer components, a simple preparation method, and good long-term storage stability;

[0058] (2) The emulsion-type mucosal vaccine preparation provided by the present invention has no obvious interaction with the antigen and has good antigen universality;

[0059] (3) The emulsion-type mucosal vaccine preparation provided by the present invention has an emulsion particle size of 100-200nm and a droplet size of 2-5μm after atomization, and is suitable for various novel immunization methods such as nasal drops, atomized inhalation, and pulmonary administration.

[0060] (4) Compared with general oil-in-water emulsions, the emulsion provided by the present invention can reduce mucus viscosity, enhance antigen adhesion, promote antigen penetration, and increase antigen uptake, playing a role in multiple processes of immune activation, thereby inducing a higher mucosal immune response and improving vaccine protective efficacy. Attached Figure Description

[0061] Figure 1 A schematic flowchart illustrating the preparation method of the emulsion-type mucosal vaccine formulation provided by the present invention;

[0062] Figure 2 This is a schematic diagram of the structure of the emulsion-type mucosal vaccine formulation provided by the present invention;

[0063] Figure 3 This is a schematic diagram of the emulsion particle size variation in Test Example 1 of the present invention;

[0064] Figure 4 This is a schematic diagram showing the distribution of antigen and emulsion in Test Example 2 of the present invention;

[0065] Figure 5 The antibody detection results after nasal immunization in Test Example 3 of the present invention include serum IgG and nasal washing / lung washing fluid sIgA levels;

[0066] Figure 6 The antibody detection results after immunization via lung administration in Test Example 4 of the present invention include serum IgG and nasal lavage / lung lavage fluid sIgA levels;

[0067] Figure 7 The survival rate curve results are for the challenge experiment of Test Example 5 of this invention. Detailed Implementation

[0068] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention will be further explained through specific embodiments below. However, the present invention is not limited to the scope of the embodiments.

[0069] like Figure 1As shown, the preparation method of the emulsion-type mucosal vaccine formulation provided by the present invention includes: first, adding a certain amount of choline hydroxide or choline bicarbonate dropwise to an aqueous solution of an acidic compound, stirring at room temperature for 24 hours to prepare a functional aqueous phase; then, weighing the prescribed amounts of the functional aqueous phase, oil phase, and surfactant according to the proportion; next, mixing the functional aqueous phase, oil phase, and surfactant, emulsifying under ultrasonic conditions, and then sterilizing using a 0.22 μm filter to prepare an emulsion; finally, mixing the emulsion with an antigen solution in a certain proportion, vortexing, and obtaining the emulsion-type mucosal vaccine formulation.

[0070] like Figure 2 As shown, the emulsion-type mucosal vaccine formulation prepared by the present invention mainly includes a functional aqueous phase, an oil phase, and a surfactant located at the oil-water interface, wherein the antigen is dispersed in the functional aqueous phase.

[0071] Example 1

[0072] The preparation method of the emulsion-type mucosal vaccine formulation in Example 1 is as follows:

[0073] (1) Weigh 0.123g of nicotinic acid and dilute it to 0.5mL with ultrapure water to prepare a nicotinic acid suspension with a concentration of 2M.

[0074] (2) Measure 0.27 mL of choline hydroxide aqueous solution (45 wt%, 3.7 M), add water to 0.5 mL, and add it dropwise to the nicotinic acid suspension. The nicotinic acid particles can be seen to gradually dissolve. Stir the reaction at room temperature for 24 h to obtain the functional aqueous phase.

[0075] (3) Weigh 1g of functional aqueous phase, 0.75g of squalene and 0.25g of Tween 80, and vortex mix them evenly;

[0076] (4) Emulsify for 2 minutes under ultrasonic conditions with a power of 300W (ultrasonic for 2 seconds, pause for 3 seconds), and then sterilize with a 0.22μm filter to prepare an emulsion;

[0077] (5) Weigh 4 mL of influenza virus subunit vaccine H1N1 antigen solution (concentration of 75 μg / mL) and 2 mL of emulsion. Vortex mix the emulsion and antigen solution to obtain emulsion-type mucosal vaccine preparation.

[0078] Example 2

[0079] The preparation method of the emulsion-type mucosal vaccine formulation in Example 2 is as follows:

[0080] (1) Weigh 0.123g of nicotinic acid and dilute it to 0.5mL with ultrapure water to prepare a nicotinic acid suspension with a concentration of 2M.

[0081] (2) Measure 0.18 mL of choline bicarbonate aqueous solution (80 wt%, 5.6 M), add water to 0.5 mL, and add it dropwise to the nicotinic acid suspension. The nicotinic acid particles can be seen to gradually dissolve. Stir the reaction at room temperature for 24 h to obtain the functional aqueous phase.

[0082] (3) Weigh 1g of functional aqueous phase, 0.75g of squalene and 0.25g of Tween 80, and vortex mix them evenly;

[0083] (4) Emulsify for 2 minutes under ultrasonic conditions with a power of 300W (ultrasonic for 2 seconds, pause for 3 seconds), and then sterilize with a 0.22μm filter to prepare an emulsion;

[0084] (5) Weigh 4 mL of influenza virus subunit vaccine H1N1 antigen solution (concentration of 75 μg / mL) and 2 mL of emulsion. Vortex mix the emulsion and antigen solution to obtain emulsion-type mucosal vaccine preparation.

[0085] Examples 3-8

[0086] The emulsion-type mucosal vaccines of Examples 3-8 were prepared using the same preparation steps and conditions as in Example 1, and the specific components are shown in Tables 1 and 2.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091]

[0092] Comparative Example 1

[0093] The emulsion vaccine formulation for mucosal immunization in Comparative Example 1 is an oil-in-water emulsion, and its preparation method is as follows:

[0094] (1) Weigh 1g of 10mM phosphate buffer (pH 8.00), 0.75g of squalene, and 0.25g of Tween 80, and vortex mix them thoroughly.

[0095] (2) Emulsify for 2 minutes under ultrasonic conditions with a power of 300W (ultrasonic for 2 seconds, pause for 3 seconds), and then sterilize with a 0.22μm filter to prepare an emulsion;

[0096] (3) Weigh 4 mL of influenza virus subunit vaccine H1N1 antigen solution (concentration of 75 μg / mL) and 2 mL of emulsion. Vortex mix the emulsion and antigen solution to obtain a common water-in-oil emulsion mucosal vaccine preparation.

[0097] Comparative Example 2

[0098] Comparative Example 2 uses an emulsion vaccine formulation for mucosal immunization, specifically the commercially available MF59 emulsion (AddaVax). TM Using Invivogen (USA) as an adjuvant, the preparation method is as follows:

[0099] Weigh 2 mL of influenza virus subunit vaccine H1N1 antigen solution (concentration of 50 μg / mL) and 2 mL of emulsion. Vortex mix the emulsion and antigen solution to obtain MF59 emulsion-type mucosal vaccine preparation.

[0100] Test Example 1

[0101] This test example demonstrates the long-term storage stability of the emulsion before it is compounded with the antigen aqueous solution. The specific method is as follows:

[0102] The emulsions corresponding to Examples 1, 5, and 7 (without being mixed with the antigen aqueous solution) were divided into several portions of 50 μL / tube and stored at 4°C. During the 12-month storage period, one sample was taken at appropriate intervals, diluted 1000 times with deionized water, and placed in a cuvette. DLS was used to detect the particle size change of the emulsion during storage. The test conditions were: temperature 24°C, three repeated measurements for each sample, 10 signals read per test, and a 3-second reading for each signal. The average particle size distribution intensity was taken as the emulsion particle size. The test results are shown below. Figure 3 As shown.

[0103] Depend on Figure 3 It is evident that after 8 months of storage, the emulsion particle size did not change significantly, demonstrating good long-term storage stability.

[0104] Test Example 2

[0105] This test case evaluates the distribution of antigen and emulsion in emulsion vaccine formulations. The specific method is as follows:

[0106] (1) Preparation of Cy5-squalene: Squalene was labeled with Cy5 dye. 10 mg of Cy5-SE dye (1 mg / mL) was added to 1 g of squalene and vortexed to obtain fluorescently labeled squalene Cy5-squalene.

[0107] (2) Preparation of FITC-H1N1: H1N1 antigen was labeled with FITC fluorescent dye. H1N1 antigen was desalted and transferred to 20 mM PB buffer (pH 8.50). FITC dye was added at F / P = 5 and incubated overnight in the dark. The protein was concentrated by centrifugation using an ultrafiltration tube and unbound dye was washed away with 1×PBS until the filtrate was clear. The concentration of labeled protein was detected by BCA method and adjusted to 75 μg / mL with 1×PBS.

[0108] (3) Preparation of Cy5-H1N1: H1N1 antigen was labeled with Cy5-SE fluorescent dye. H1N1 antigen was desalted and the medium was changed to 20 mM PB buffer (pH 8.50). Cy5-SE fluorescent dye was added at F / P = 5 and incubated overnight in the dark. The protein was concentrated by centrifugation using an ultrafiltration tube and unbound dye was washed away with 1×PBS until the filtrate was clear. The protein concentration after labeling was detected by BCA method and adjusted to 75 μg / mL with 1×PBS.

[0109] (4) The distribution of antigen and emulsion was detected using super-resolution laser confocal microscopy. Labeled FITC-H1N1 was used instead of the influenza H1N1 antigen solution (concentration 75 μg / mL) in Example 1, and labeled Cy5-squalene was used instead of the squalene in Example 1 to prepare a mucosal preparation with dual labeling of antigen and emulsion. The distribution of antigen and emulsion was detected using super-resolution laser confocal microscopy, and the results are as follows: Figure 4 As shown, green represents the antigen and red represents the emulsion.

[0110] Depend on Figure 4 As can be seen, the antigen and emulsion in Example 1 do not exhibit co-localization and do not interact with each other in the formulation, existing independently. The emulsion-type mucosal formulation has good antigen versatility.

[0111] Test Example 3

[0112] This test case evaluates the efficacy of an emulsion vaccine formulation for nasal administration of influenza H1N1 lysate antigen. The specific method is as follows:

[0113] Female Balb / c mice aged 6–8 weeks were selected and acclimatized to their environment in an SPF-grade animal facility for 5 days. They were then divided into four groups of six mice each, corresponding to Example 1, Comparative Example 1, Comparative Example 2, the nasal drop control group (antigen solution administered via direct nasal drop), and the intramuscular injection control group (antigen solution administered via direct intramuscular injection), respectively. Mice were anesthetized with isoflurane before nasal drops. Each mouse received 20 μL of nasal drop solution, 10 μL in each nostril. Vaccination was performed on days 0 and 14. Blood was collected from the orbital sinus and serum separated on days 14 and 28 after the first vaccination. Twenty-eight days after the first vaccination, the mice were euthanized, and the trachea was dissected and exposed. A V-shaped incision was made in the trachea, and 400 μL of 1×PBS was used to flush the trachea towards the nasal cavity. The collected nasal wash was used to flush the nasal cavity four times to obtain nasal wash solution. The levels of specific IgG antibodies in serum, specific IgA (sIgA) in nasal lavage fluid, and specific IgA (sIgA) in bronchoalveolar lavage fluid were measured using enzyme-linked immunosorbent assay (ELISA). The test data are shown in Table 3. Figure 5 As shown, where, Figure 5The horizontal axis in the figure corresponds to the nasal drop control group for "pure antigen nasal drops", "MF59 adjuvant nasal drops" corresponds to ratio 2, "ordinary oil-in-water nasal drops" corresponds to ratio 1, "emulsion nasal drops prepared from nicotinic acid" corresponds to Example 1, and "pure antigen intramuscular injection" corresponds to the intramuscular injection control group.

[0114] Table 3

[0115]

[0116] From Table 3 and Figure 5 The results show that, during the nasal immunization process, the emulsion-type mucosal vaccine formulation of Example 1 of the present invention produced an IgG antibody titer of 109333, a nasal wash sIgA antibody titer of 42, and a lung lavage fluid sIgA antibody titer of 102. Compared with Comparative Example 1, the IgG antibody titer of the vaccine formulation of Example 1 increased by 4.31 times, the nasal wash sIgA antibody titer increased by 2.25 times, and the lung lavage fluid sIgA antibody titer increased to 102. Compared with Comparative Example 2, the IgG antibody titer of the vaccine formulation of Example 1 increased by 7.81 times, the nasal wash sIgA antibody titer increased by 2.07 times, and the lung lavage fluid sIgA antibody titer increased by 8.72 times. Compared with the intramuscular injection control group, the IgG antibody titer of the vaccine formulation of Example 1 was slightly lower, but the sIgA antibody titers in the nasal wash and lung lavage fluid were significantly increased, effectively activating the body's mucosal immunity. The above results all demonstrate that the emulsion-type mucosal vaccine formulation involved in this invention exhibits excellent mucosal immune effects when administered via nasal drops.

[0117] Test Example 4

[0118] This test case evaluates the pulmonary immunogenicity of an emulsion vaccine formulation administered to the lungs using the H1N1 influenza split antigen. The specific method is as follows:

[0119] Female Balb / c mice aged 6–8 weeks were selected and, after acclimatizing to their environment in an SPF-grade animal facility for 5 days, were divided into four groups of six mice each: the PBS group, Example 3, the lung control group (direct lung administration of antigen solution), and the intramuscular injection control group (direct intramuscular injection of antigen solution). Mice were deeply anesthetized with afodin, and each mouse received 25 μL of the drug in the lungs. Vaccination was performed on days 0 and 14, and blood was collected from the orbital sinus and serum separated on days 14 and 28 after the first vaccination. Twenty-eight days after the first vaccination, the mice were euthanized, and the trachea was dissected and exposed. A V-shaped incision was made in the trachea, and 400 μL of 1×PBS was used to flush from the trachea towards the nasal cavity. The collected nasal wash was used to flush repeatedly four times to obtain nasal wash fluid. Lung lavage fluid was obtained by flushing from the trachea towards the lungs with 1000 μL of 1×PBS, repeatedly pipetting four times until the liquid was homogeneous, and then aspirating the lavage fluid. The levels of specific IgG antibodies in serum, specific IgA (sIgA) in nasal lavage fluid, and specific IgA (sIgA) in bronchoalveolar lavage fluid were measured using enzyme-linked immunosorbent assay (ELISA). The test data are shown in Table 4. Figure 6 As shown, where, Figure 6 The horizontal axis “pure antigen lung” corresponds to the lung control group, “pure antigen intramuscular injection” corresponds to the intramuscular injection control group, and “nasal drops of emulsion prepared from nicotinic acid” corresponds to Example 3.

[0120] Table 4

[0121]

[0122] From Table 4 and Figure 6 The results show that, during pulmonary immunization using the emulsion-type mucosal vaccine formulation of Example 3 of the present invention, the IgG antibody titer was 226,667, the nasal wash sIgA antibody titer was 20, and the lung lavage fluid sIgA antibody titer was 333. Compared with the intramuscular injection control group, the IgG antibody titer of the vaccine formulation of Example 3 was comparable, while the nasal wash sIgA antibody titer increased to 20 and the lung lavage fluid sIgA antibody titer increased to 333. Compared with the lung control group, the IgG antibody titer of the vaccine formulation of Example 3 increased by 17 times, the nasal wash sIgA antibody titer increased to 20, and the lung lavage fluid sIgA antibody titer increased by 194.74 times, demonstrating a significant mucosal immune advantage. All the above results prove that the emulsion vaccine formulation involved in the present invention exhibits excellent mucosal immune effects via pulmonary immunization.

[0123] Comparative analysis of the results of Test Examples 3 and 4 revealed that the emulsion vaccine formulation of this invention achieved good results in mucosal immunization methods such as nasal drops and pulmonary administration. Furthermore, compared to nasal administration (33% v / v emulsion, IgG titer 109333, sIgA titer 42), the pulmonary administration formulation, using a lower emulsion concentration (approximately 6% emulsion), showed an approximately 2.1-fold increase in IgG titer and an increase in sIgA titer in bronchoalveolar lavage fluid to 333, demonstrating significant application potential.

[0124] Test Example 5

[0125] This test case evaluates the protective efficacy of an emulsion vaccine formulation against H1N1 influenza virus in a challenge experiment. The specific method is as follows:

[0126] Female Balb / c mice aged 6–8 weeks were selected and acclimatized to the environment in a P2-level laboratory for 5 days. They were then divided into four groups of eight mice each, corresponding to the PBS group, Example 1, Example 3, and the intramuscular injection control group (direct intramuscular injection of antigen solution). Mice were deeply anesthetized with afodin. The antigen immunization dose was 2 μg / mouse. In the PBS group and Example 1 group, the dose was 20 μL intranasally (10 μL in each nostril). In the Example 3 group, the dose was 25 μL intrapulmonaryly. In the intramuscular injection control group, each mouse received an intramuscular injection of 50 μL. Vaccination was performed on days 0 and 14, and on day 28, the H1N1 strain (A / Victoria / 4897 / 2022) was administered intranasally at a volume of 50 μL, with a challenge dose of 30 × LD50. Body weight was monitored daily for 7 days post-inoculation. Mice were considered dead when their body weight decreased to 75% of their initial weight. Test data are shown in Table 5. Figure 7 As shown, where, Figure 7 The “lung administration group” corresponds to Example 3, and the “nasal administration group” corresponds to Example 1.

[0127] Table 5

[0128] Group Emulsion type Survival rate (%) 7 days after viral infection PBS group / 0 Example 1 Functional emulsion 50 Example 3 Functional emulsion 100 Intramuscular injection group / 0

[0129] From Table 5 and Figure 7 The results showed that even under high challenge doses (30×LD50), both Example 1 and Example 3 maintained significantly better survival rates than the intramuscular injection group. The survival rate of mice in the intramuscular injection group was 0%, the survival rate of mice immunized by intranasal drops of the vaccine formulation in Example 1 was 50%, and the survival rate of mice after lung administration of the vaccine formulation in Example 3 was 100%.

[0130] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing an emulsion-type mucosal vaccine formulation, characterized in that, Includes the following steps: (1) A certain amount of choline hydroxide or choline bicarbonate solution was added dropwise to an aqueous solution of an acidic compound, and the mixture was stirred at room temperature for 24 hours to prepare a functional aqueous phase. (2) Weigh out the prescribed amounts of functional aqueous phase, oil phase, and surfactant according to the proportions; (3) The functional aqueous phase, oil phase and surfactant are mixed and emulsified under ultrasonic conditions, and then sterilized by a 0.22μm filter to prepare an emulsion; (4) Mix the emulsion and the antigen solution in a certain proportion and vortex to obtain an emulsion-type mucosal vaccine preparation.

2. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The acidic compound includes any one of glycolic acid, lactic acid, nicotinic acid, geranilic acid, and glycine.

3. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The molar ratio of choline hydroxide or choline bicarbonate to the acidic compound is 1:0.5-1:

2.

4. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The oil phase includes any one or a combination of at least two of squalene, soybean oil, isopropyl myristate, or tocopherol.

5. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The surfactant includes any one or a combination of at least two of the following: nonionic surfactants alkyl glucoside, fatty acid ester, Span, Tween, and ionic surfactants stearic acid, sulfated castor oil, and oleic acid.

6. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The functional aqueous phase has a molar concentration of 0.5-5M and an addition amount of 50%-70% (w / w), the oil phase has an addition amount of 5%-45% (w / w), and the surfactant has an addition amount of 5%-25% (w / w).

7. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The ultrasonic power is 100-500w, and the emulsification time is 30s-5min. During the ultrasonic process, the operation is paused for 3 seconds after every 2 seconds of ultrasonic operation.

8. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The antigens are disease-related antigens transmitted via the respiratory tract, including any one or a combination of at least two of the following: coronavirus vaccine antigen, novel coronavirus vaccine antigen, influenza virus vaccine antigen, parainfluenza virus vaccine antigen, respiratory syncytial virus vaccine antigen, human metapneumovirus vaccine antigen, adenovirus vaccine antigen, measles virus vaccine antigen, herpes zoster virus vaccine antigen, pneumococcal vaccine antigen, Bordetella pertussis vaccine antigen, Mycobacterium tuberculosis, foot-and-mouth disease vaccine antigen, and Mycoplasma pneumoniae vaccine antigen.

9. The method for preparing an emulsion-type mucosal vaccine formulation according to claim 1, characterized in that, The antigens include any one or a combination of at least two of the following: live attenuated virus vaccine antigens, inactivated virus vaccine antigens, split vaccine antigens, virus-like particle vaccine antigens, subunit vaccine antigens, polysaccharide conjugate vaccine antigens, nucleic acid vaccine antigens, or synthetic peptide vaccine antigens.

10. The use of the emulsion-type mucosal vaccine formulation according to any one of claims 1-9 in the development of novel mucosal vaccines.

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