Vaccine composition as well as preparation method and application thereof

Abstract

The invention discloses a vaccine composition and a preparation method thereof, the vaccine composition comprises a first component and a second component, the first component is a recombinant protein antigen, the second component is an oil-in-water composite adjuvant, and the oil-in-water composite adjuvant comprises a water phase, an oil phase, an emulsifier, a Toll-like receptor stimulant and / or an STING stimulant. The vaccine composition provided by the invention can stimulate a body to generate a high-level specific IgG antibody and a neutralizing antibody, and has a good cellular immune effect at the same time. The second component provided by the invention is simple in preparation process, low in cost, stable in dosage form, easy to store and good in immune effect, and has a wide application prospect in vaccine or drug development.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to a vaccine composition, a preparation method thereof, and uses thereof in the fields of immunotherapy and prevention. Background Art

[0002] As a biological product, vaccines play an irreplaceable and critical role in the prevention and treatment of diseases. The immunogenicity of some vaccines is generally weak, and adjuvants are usually added to improve the immunogenicity of vaccines. Adjuvants are a class of substances that can enhance or change the body's specific immune response to antigens. They are non-immunogenic themselves and will not cause the body's immune response. Adjuvants can effectively reduce the antigen content or dosage contained in vaccine products. At the same time, they can effectively activate the humoral and cellular immune systems, induce stronger protective responses, and thus improve the effectiveness of vaccines. However, only a few adjuvants have been approved for clinical use. Therefore, the development of stable, safe, and efficient vaccine adjuvants is of great significance to the rapid construction and clinical transformation of new vaccine adjuvants.

[0003] Oil-emulsion adjuvants are widely used commercial vaccine adjuvants, and water-in-oil adjuvants are one type of adjuvants. They have high stability, high cellular uptake rate and high biosafety, and can effectively co-deliver antigens and adjuvants. Water-in-oil adjuvants are usually prepared by high-pressure homogenization, ultrasound, microfluidizer and other methods, and surfactants (such as Tween 80 and Span 85, etc.) are added to stabilize the emulsion. The mechanism of action of water-in-oil adjuvants is to activate immune cells at the injection site, induce monocytes, neutrophils, eosinophils and DC cells to be rapidly recruited to the injection site, and then enter the draining lymph nodes to produce more antigen-presenting cells, promote the secretion of chemokines and cytokines, thereby enhancing antigen uptake and immune activation. However, there is currently no water-in-oil vaccine adjuvant with stable performance on the market. In addition, the water-in-oil adjuvants in the prior art still have defects such as low immune efficacy in the use process due to the unreasonable formulation ingredients and preparation process. In addition, the raw materials of the oil phase components used in the prior art oil-in-water adjuvants, such as squalene, are expensive and difficult to obtain. Therefore, there is an urgent need to develop a new oil-in-water adjuvant with good effect and low cost as an alternative. Summary of the Invention

[0004] Technical issues solved:

[0005] The purpose of this disclosure is to provide a vaccine composition that is safe, has a good immune effect, is simple to manufacture, is low in cost, and can rapidly induce the body to produce high levels of specific IgG antibodies and neutralizing antibodies, while also having a good cellular immune effect. The present invention has broad application prospects in the development of vaccines or drugs. The technical solutions provided are:

[0006] A vaccine composition comprises a first component and a second component, wherein the first component is a recombinant protein antigen, and the second component is an oil-in-water composite adjuvant, wherein the oil-in-water composite adjuvant comprises an aqueous phase, an oil phase, an emulsifier, and also comprises a Toll-like receptor agonist and / or an interferon gene stimulator.

[0007] In some embodiments of the present disclosure, the vaccine is an inactivated vaccine, a live attenuated vaccine, a genetically engineered vaccine, a subunit vaccine, a genetically reassortant vaccine, a DNA vaccine, or an mRNA vaccine formulation. In the present invention, preferably, the first component is a genetically engineered vaccine.

[0008] In some embodiments of the present disclosure, the oil-in-water composite adjuvant comprises 0.5 wt% to 1 wt% emulsifier, 5 wt% to 10 wt% oil phase, and further comprises a Toll-like receptor agonist with a final concentration of 0.5 to 5 mg / ml and / or a STING agonist with a final concentration of 100 to 300 μg / ml.

[0009] In some embodiments of the present disclosure, the buffer of the aqueous phase may be one or more selected from citric acid / citrate buffer, phosphate buffer, carbonate buffer, acetate buffer, borate buffer, histidine buffer, tris(hydroxymethyl)methane buffer or barbiturate buffer.

[0010] In some embodiments of the present disclosure, the aqueous phase buffer comprises a final concentration of 0.01 to 1000 mM histidine and a final concentration of 0.01 to 1000 mM NaCl; the buffer has a pH range of 6.5 to 7.0. In the present invention, preferably, the buffer is 10 mM histidine-150 mM NaCl, with a pH of 6.8. In some embodiments of the present disclosure, the oil phase may include one or more selected from soybean oil, peanut oil, fish oil, olive oil, sunflower oil, cottonseed oil, sesame oil, corn oil, or coconut oil.

[0011] In some embodiments of the present disclosure, the specific composition requirements of the above-mentioned soybean oil, peanut oil, fish oil, olive oil, sunflower oil, cottonseed oil, sesame seed oil, corn oil or coconut oil comply with the records in the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0012] In some embodiments of the present disclosure, the emulsifier may include sorbitan fatty acid ester and / or a nonionic surfactant.

[0013] In some embodiments of the present disclosure, the sorbitan fatty acid ester may be one or more selected from Span 20, Span 40, Span 60, Span 80, or Span 85.

[0014] In some embodiments of the present disclosure, the nonionic surfactant may be one or more selected from Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Tween 81, or Tween 85. In some embodiments of the present disclosure, the oil-in-water composite adjuvant may further comprise an immunomodulator, such as one or more selected from Toll-like receptor agonists, STING agonists, BCG polysaccharide nucleic acids, and mannan peptides. The Toll-like receptor agonist may be selected from one or more of TLR3, TLR7, TLR8, and TLR9, preferably TLR3, and more preferably poly I-C. The STING agonist may be selected from one or more of cyclic dinucleotides, aminobenzimidazoles, anthrones, benzothiophenes, and benzodienes, preferably cyclic dinucleotides.

[0015] In some embodiments of the present disclosure, the method for preparing the oil-in-water composite adjuvant may include the following steps:

[0016] Step 1) adding the Toll-like receptor agonist and / or STING agonist to a buffer solution to prepare solution I;

[0017] Step 2) adding the oil phase and the emulsifier to the above solution I and stirring to obtain solution II;

[0018] Step 3) High-pressure homogenization of the above solution II was performed at 1000-2000 bar for 6 times to prepare solution III;

[0019] Step 4) The solution III is filtered and sterilized to finally prepare a solution IV, which is the oil-in-water composite adjuvant.

[0020] In some embodiments of the present disclosure, the particle size of the oil-in-water composite adjuvant may be 190 to 220 nm, the PdI value may be 0.09 to 0.18, and the particle uniformity may be good. For example, in some embodiments of the present disclosure, the particle size of the oil-in-water composite adjuvant can be about 190 nm, about 191 nm, about 192 nm, about 193 nm, about 194 nm, about 195 nm, about 196 nm, about 197 nm, about 198 nm, about 199 nm, about 200 nm, about 201 nm, about 202 nm, about 203 nm, about 204 nm, about 205 nm, about 206 nm, about 207 nm, about 208 nm, about 209 nm, about 210 nm, about 211 nm, about 212 nm, about 213 nm, about 214 nm, about 215 nm, about 216 nm, about 217 nm, about 218 nm, about 219 nm, or about 220 nm. The PdI value of the oil-in-water composite adjuvant can be about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, or about 0.18.

[0021] Another aspect of the present disclosure provides a vaccine comprising the above-mentioned oil-in-water composite adjuvant, and an antigen or a nucleotide encoding the antigen.

[0022] In some embodiments of the present disclosure, the vaccine may be a vaccine against viruses, bacteria, mycoplasmas, rickettsiae, spirochetes, fungi, parasites, etc.

[0023] In some embodiments of the present disclosure, the virus may be a herpesviridae, poxviridae, adenoviridae, human papillomaviridae, parvoviridae, reoviridae, togaviridae, flaviviridae, coronaviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, filoviridae, retroviridae, picornaviridae, astroviridae, caliciviridae, or hepatotropic virus.

[0024] In some embodiments of the present disclosure, the virus can also be herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus, smallpox virus, human adenovirus, human papillomavirus, parvovirus, Kabovirus, such as rotavirus, rubella virus, epidemic encephalitis B virus, dengue virus, yellow fever virus, SARS coronavirus, MERS coronavirus, new coronavirus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rabies virus, Hantavirus, Xinjiang Crimean hemorrhagic fever virus, Ebola virus, Marburg virus, HIV, enterovirus, coxsackievirus, echovirus, poliovirus, astrovirus, norovirus, hepatitis B virus, hepatitis C virus, hepatitis A virus, and hepatitis E virus.

[0025] Beneficial effects:

[0026] The vaccine composition provided by the present disclosure can stimulate the body to produce high levels of specific IgG antibodies and neutralizing antibodies, while also having a good cellular immune effect. The second component of the present invention, an oil-in-water composite adjuvant, has a simple preparation process, low cost, a stable dosage form, easy storage, and a good immune effect, and has broad application prospects in vaccine or drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the particle size and polydispersity coefficient of the oil-in-water composite adjuvant prepared in the examples disclosed herein;

[0028] Figure 2 This is a diagram showing the detection results of specific IgG antibodies and neutralizing antibodies in the embodiments of the present disclosure;

[0029] Figure 3 This is a diagram showing the results of cellular immunity level detection in the embodiments of the present disclosure;

[0030] Figure 4 These are photos of five types of prescribed fish oil emulsions in the examples disclosed herein;

[0031] Figure 5 The results of particle size and polydispersity coefficient in the embodiments of the present disclosure are shown in FIG.

[0032] Figure 6 This is a graph showing electron microscopy results in the embodiments of the present disclosure;

[0033] Figure 7 This is a diagram showing the results of animal experiments in the embodiments of the present disclosure;

[0034] Figure 8 This is a diagram showing the results of animal experiments in the embodiments of the present disclosure;

[0035] Figure 9 Graph showing the results of animal experiments in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0036] The present invention discloses a vaccine composition. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention, and relevant persons can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0037] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The terms "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example" etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0038] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common terms in molecular biology can be found in Lewin's GENE XII, Jocelyn E. Krebs / Elliott S. Goldstein / Stephen T. Kilpatrick, published by Jones & Bartlett, 2018.

[0039] The terms "adjuvant," "vaccine adjuvant," and "immunoadjuvant" as used herein refer to a class of substances that can non-specifically bind to or be mixed with an antigen / immunogen / immunogenic substance to enhance the immunogenicity and immunoprotective effects of the antigen / immunogen / immunogenic substance, i.e., enhance humoral and / or cellular immune responses, without being immunogenic themselves. They can also be understood as "a substance that, when combined with an antigen, can produce a more effective immune response than the antigen alone."

[0040] Poly (I:C), also known as poly (I:C), is a synthetic double-stranded RNA. It is an interferon inducer with antiviral and immunomodulatory properties.

[0041] In the present disclosure, the subject of administration of the "adjuvant", "vaccine adjuvant", "immunoadjuvant", non-specific immune response enhancer, etc. is any mammal, including but not limited to domestic animals and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats or rodents, etc.), primates and humans (Homo sapiens). Preferably, the mammal is a human.

[0042] In certain embodiments of the present disclosure, the type of vaccine may include, for example, an inactivated vaccine, a live attenuated vaccine, a protein vaccine, a bacterial polysaccharide and polysaccharide protein conjugate vaccine, a genetically engineered vaccine, or a genetically reassorted vaccine. Examples of the inactivated vaccine include whole virus vaccines or split-type vaccines, for example, inactivated polio vaccine, diphtheria and tetanus vaccine, split influenza vaccine, inactivated hepatitis A vaccine, meningococcal polysaccharide vaccine, etc. The live attenuated vaccine is made from treated pathogens that have lost their pathogenicity but are still able to induce an immune response. The preparation method of this vaccine is to reduce or eliminate the pathogenicity of the pathogen by physical or chemical methods, or by continuous passage culture under in vitro conditions, but maintain good immunogenicity. For example, measles, mumps and rubella (MMR) vaccine, varicella vaccine, yellow fever vaccine, rotavirus vaccine, oral polio vaccine (OPV), etc. Protein vaccines are made from specific pathogen proteins that can induce an immune response. The preparation method of this vaccine is to directly deliver specific proteins of the pathogen and adjuvants that stimulate the immune response into human cells. For example, a recombinant new coronavirus protein vaccine. The bacterial polysaccharide and polysaccharide-protein conjugate vaccine is composed of bacterial polysaccharide antigens combined with a protein carrier. This vaccine is prepared by chemically coupling specific bacterial polysaccharides with a protein carrier, enabling the polysaccharide to be recognized by T cells, thereby triggering a stronger immune response. Examples include Haemophilus influenzae polysaccharide conjugate vaccine, meningococcal polysaccharide conjugate vaccine, and pneumococcal polysaccharide conjugate vaccine. Genetically engineered vaccines are vaccines prepared using genetic engineering techniques. This vaccine is prepared by cloning the pathogen's specific antigen gene into an expression vector, then inducing expression of the exogenous protein using a prokaryotic or eukaryotic expression system, and then producing the vaccine through purification and other process steps. Examples include recombinant hepatitis B vaccine, BCG vaccine, and DNA vaccine. Genetically reassorted vaccines are produced using recombinant microorganisms obtained through genetic reassortment. Typically, a weak strain that is non-pathogenic to humans is co-infected with a virulent strain (mostly a wild-type strain). Genomic segments are exchanged between the weak and wild-type strains, resulting in reassortment. Specific methods are then used to screen for reassorted strains that are non-pathogenic to humans but contain immunogenic gene segments from the wild-type strain. For example, rotavirus vaccine, influenza virus vaccine, etc. In one embodiment of the present disclosure, the vaccine may be a protein vaccine or an inactivated vaccine.

[0043] antigen:

[0044] The antigen in the first component of this example is a recombinant protein RBD antigen against the novel coronavirus (SARS-CoV-2). This recombinant coronavirus protein RBD antigen is an intramolecular trimer composed of the RBD sequences of three Omicron variants of the novel coronavirus, BQ.1.1, BA.2.3.20, and XBB, in end-to-end sequence. The amino acid sequence is shown in SEQ ID No. 1. This protein is recombinantly expressed in CHO cells and purified by a series of chromatography.

[0045] The recombinant protein RBD antigen in this embodiment is a new fusion protein designed based on the structural characteristics of the RBD region of the new coronavirus S protein using computational biology methods. The protein contains three RBD domains, which can form a trimer form with a stable antigen conformation without introducing any exogenous connecting arms or other irrelevant components, thereby realizing RBD protein trimerization. After recombinant expression and purification of the RBD trimer protein using genetic engineering technology, it is mixed with an adjuvant to prepare a vaccine. After immunization at a certain dose and dosage, protective neutralizing antibodies against a variety of new coronavirus epidemic strains can be produced for the treatment and / or prevention of SARS-CoV-2 infection and / or new coronavirus disease (COVID-19). Because the RBD region has a clear function and a clear structure, it is responsible for recognizing the angiotensin-converting enzyme 2 receptor of the host cell. At the same time, the antibodies produced against RBD have clear functions and are target-specific, which can avoid inducing the body to produce antibody-dependent enhancement (Antibody Dependent Enhancement, ADE) to the greatest extent.

[0046] vaccine:

[0047] The term "vaccine" as used herein refers to a product that, when administered to a human or animal, induces an immune response, including humoral and / or cellular immunity. Vaccines can be prophylactic or therapeutic. Vaccines include an immunogenic substance and, optionally, at least one immunomodulator. In certain embodiments of the present disclosure, the immunogenic substance is a portion of a viral protein, such as SARS-CoV-2. SARS-CoV-2 belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae, genus Betacoronavirus, subgenus Sarbecovirus, and species SARS-like. It is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 29.9 kb. The vast majority of the genome encodes nonstructural proteins involved in viral replication and translation. A small portion of the sequence encodes structural proteins, such as the spike protein (S), membrane protein (M), envelope protein (E), and nucleoprotein (N). In addition, there are several accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14, all of which are involved in viral assembly. The S, M, and E proteins constitute the viral envelope and are the primary surface antigens that elicit an immune response. The S protein is a transmembrane glycoprotein with a molecular weight of approximately 150 kDa that forms a prominent homotrimer on the viral surface. The S protein is composed of two functional subunits, which are cleaved at the boundary between the S1 and S2 subunits (the S1 / S2 cleavage site). The two subunits remain non-covalently associated in their pre-fusion conformation. The S2 subunit is also composed of multiple domains, and its primary function is to mediate fusion between the virus and the host cell. The distal S1 subunit is structurally divided into four distinct domains: the NTD, RBD, CTD1, and CTD2. The RBD is the receptor-binding domain, primarily responsible for binding to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2), thereby mediating viral infection. Therefore, the S protein and RBD are currently the main targets for genetically engineered vaccine development. Another example is the H3N2 influenza virus. H3N2 is a subtype of influenza A virus and a major cause of human influenza epidemics. The virus is named after the two proteins on its surface: hemagglutinin (HA) and neuraminidase (NA). H3N2 can exchange genes for internal proteins through genetic recombination.

[0048] Oil-in-water adjuvants:

[0049] Oil-in-water emulsion (also known as O / W emulsion) is a special emulsion in which the oil phase is dispersed in the water phase to form an emulsion. It is composed of an aqueous phase, an oil phase, and an emulsifier. Its use as a vaccine adjuvant can enhance the immune response effect of immunogenic substances. For example, AS01 oil-in-water emulsion has been widely studied and used in vaccine development. AS01 is an emulsion formed by squalene oil in an aqueous phase, which contains the immunostimulants MPL and QS-21. AS03 oil-in-water emulsion is also used as an adjuvant for influenza vaccines (such as Prepandrix and Q-PanH5N1). AS03 is an emulsion formed by squalene oil in an aqueous phase, and the immunostimulant α-tocopherol is added.

[0050] The emulsifier in an oil-in-water emulsion generally accounts for about 20% of the total oil phase. In oil-in-water emulsions, the commonly used emulsifiers are anionic and nonionic surfactants, whose HLB values are generally between 8 and 18, with nonionic types being the majority.

[0051] Preparation method:

[0052] In embodiments of the present disclosure, a suitable dispersion buffer system can be selected. The term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of the pharmaceutical formulation. Suitable pharmaceutically acceptable buffers include, but are not limited to, citric acid / citrate buffer, phosphate buffer, carbonate buffer, acetate buffer, borate buffer, histidine buffer, tris buffer, succinate buffer, or barbiturate buffer. In certain embodiments, the concentration of the buffer is about 0.01 mM to about 1000 mM, about 0.1 mM to about 1000 mM, about 0.1 mM to about 500 mM, about 0.1 mM to about 200 mM, about 0.1 mM to about 100 mM, about 1 mM to about 1000 mM, about 1 mM to about 500 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 2 mM to about 60 mM, about 4 mM to about 60 mM, or about 4 mM to about 40 mM, about 5 mM to about 20 mM, or about 5 mM to about 25 mM. In order to achieve better results, in certain embodiments of the present disclosure, histidine-HCl is selected, which comprises HIS-Cl and 150 mM NaCl at final concentrations of 10 mM, respectively; the pH value of the histidine-HCl buffer is 6.5-7.0.

[0053] The term "high-pressure homogenization" is a method of reducing particle size by adding target particles to a device that combines pressure and mechanical force to decompose the target particles. The mechanical forces used in high-pressure homogenization may include impact, shearing, and cavitation, etc. Commonly used equipment, for example, a high-pressure homogenizer. In one embodiment of the present disclosure, the particle size of the solid particles after homogenization can be 190 to 220 nm, and the PdI value can be 0.09 to 0.18, at which point the effect is optimal. The method for measuring particle size is known in the prior art. For example, dynamic light scattering (DLS), transmission electron cryomicroscopy (TEM), nanoparticle tracking analysis (NTA), etc.

[0054] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0055] Example:

[0056] The recombinant novel coronavirus protein RBD antigen used in the first component of this embodiment is an intramolecular trimer composed of the RBD sequences of three Omicron variants of the novel coronavirus BQ.1.1, BA.2.3.20 and XBB connected in end-to-end sequence. The amino acid sequence is shown in SEQ ID No. 1. The protein is recombinantly expressed in CHO cells and purified by a series of chromatography.

[0057]

[0058]

[0059] Example 1: Preparation of an oil-in-water composite adjuvant using soybean oil as the oil phase

[0060] The preparation method of the oil-in-water composite adjuvant with immune enhancement effect prepared by using soybean oil as the oil phase comprises the following steps:

[0061] a) Add Poly(I:C) to a final concentration of 1 mg / ml to the histidine-HCl buffer solution to prepare Solution I;

[0062] b) 5% (w / v) soybean oil, 0.5% (w / v) Tween 80, and 0.5% (w / v) Span 85 were added to Solution I and stirred on a magnetic stirrer at 400 rpm for 10 min to prepare Solution II.

[0063] c) High-pressure homogenization of Solution II at 1000-2000 bar for 6 times to prepare Solution III;

[0064] d) Filter solution III through a 0.22 μm filter to sterilize and finally prepare solution IV, which is an oil-in-water composite adjuvant. The particle size of solution IV is measured by dynamic light scattering. The results are as follows: Figure 1 As shown, the particle size is 213.4 nm, the PdI value is 0.125, and the particle uniformity is good.

[0065] Example 2: Effect of an oil-in-water composite adjuvant with soybean oil as the oil phase on the humoral immune effect of a recombinant novel coronavirus antigen protein

[0066] BALB / c mice of SPF grade at 6 to 8 weeks of age were divided into 10 mice per group and immunized in groups according to the animal experimental scheme shown in Table 1. The recombinant novel coronavirus antigen protein was mixed with different adjuvants to prepare immune substances. Two intramuscular immunizations were performed at 0 week and 2 week. Blood was collected from the intracanthal vein at 3 week, and serum was separated and inactivated at 56°C for 30 minutes. The neutralizing antibody titer of serum against BA.1 novel coronavirus was detected by pseudovirus microneutralization test, and the IgG2a and IgG1 antibody levels in serum were detected by ELISA test. The test results are as follows: Figure 2 and as shown in Table 2.

[0067] Table 1. Animal experiment protocol

[0068]

[0069] Table 2. Results of mouse serum specific IgG antibody and neutralizing antibody detection

[0070] Group Pseudovirus (GMT) IgG1(GMT) IgG2a (GMT) 1 20 50 50 2 20 50 50 3 81 298283 5619 4 1661 1666304 115314

[0071] Example 3: Effect of an oil-in-water composite adjuvant with soybean oil as the oil phase on the cellular immune effect of recombinant novel coronavirus antigen protein

[0072] BALB / c mice of SPF grade at 6 to 8 weeks of age were divided into 10 mice per group and immunized according to the animal experimental scheme shown in Table 3. The recombinant novel coronavirus antigen protein was mixed with different adjuvants to prepare immune substances. Three intramuscular injections were performed at 0 wk, 2 wk, and 4 wk. The spleens of the mice were collected at 6 wk, and the spleen mononuclear cells of the mice were isolated. The cellular immunity levels of the mice, IFNγ, IL-5, and IL-17A, were detected by ELISPOT assay. The cellular immunity test results are shown in the figure. Figure 3 and as shown in Table 4.

[0073] Table 3. Animal experiment plan

[0074]

[0075] Table 4. Cellular immunity test results

[0076] Group IFNγ IL-5 IL-17A 1 1 4 6 2 583 268 40 3 836 705 13

[0077] Example 4: Preparation of an oil-in-water composite adjuvant with fish oil as the oil phase

[0078] The preparation method of the oil-in-water composite adjuvant with immune enhancement effect prepared by using fish oil as the oil phase comprises the following steps:

[0079] a) Poly(I:C) was added to the histidine-hydrochloric acid buffer solution to final concentrations of 0 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml, respectively. The specific formula is shown in Table 5 to prepare Solution I;

[0080] b) adding fish oil at a fixed content of 5% (w / v), Tween 80 at a fixed content of 0.5% (w / v), and Span 85 at a fixed content of 0.5% (w / v) to Solution I, and stirring on a magnetic stirrer at 400 rpm for 10 minutes to prepare Solution II;

[0081] Table 5. Fish oil emulsion formula

[0082]

[0083] c) high-pressure homogenization of solution II at 1000-2000 bar for 6 times to prepare solution III;

[0084] d) Filter solution III through a 0.22 μm filter for sterilization to prepare solution IV, which is an oil-in-water composite adjuvant.

[0085] The preparation results are as follows Figure 4 As shown in FIG, the fish oil emulsions prepared above all have the appearance of milky white fluid liquid, uniform and without stratification; the pH value is 6.7-6.9. The particle size and polydispersity coefficient of the emulsions were measured by dynamic light scattering, and the particle uniformity was good. Figure 5 Shown are the particle size and polydispersity coefficient of fish oil emulsion of Formula No. 3. Figure 6 Shown are the electron microscopy results of fish oil emulsion of formula No. 3.

[0086] Example 5: Effects of five formulations on the immune effects of recombinant novel coronavirus antigen protein

[0087] BALB / c mice of SPF grade at 6 to 8 weeks of age were divided into 10 mice per group. According to the animal experimental scheme shown in Table 6, the recombinant novel coronavirus antigen protein was mixed with different preparations to prepare immune substances. Two intramuscular immunizations were performed at 0 weeks and 2 weeks. Blood was collected from the intracanthal vein at 3 weeks. The serum was separated and inactivated at 56°C for 30 minutes. The neutralizing antibody titer of the serum against the BA.1 novel coronavirus was detected by live virus microneutralization test. The neutralizing antibody titer of the serum against the BA.1 novel coronavirus was detected by pseudovirus microneutralization test, as well as the mouse serum-specific IgG1 and IgG2a antibodies. The test results are as follows: Figure 7 and as shown in Table 7. The results show that the five formulations disclosed herein can stimulate the body to produce higher levels of specific IgG antibody titers and neutralizing antibody titers.

[0088] Table 6. Animal immunization schemes for different formulations

[0089]

[0090]

[0091] Table 7. Results of mouse serum specific IgG antibody and neutralizing antibody detection

[0092] Group IgG1(GMT) IgG2a (GMT) Wild virus (GMT) Pseudovirus (GMT) 1 50 50 10 20 2 2483350 6400 1237 1136 3 204800 1600 67 77 4 620838 3200 463 502 5 2017107 77605 1505 2407 6 3985480 109750 1405 2493 7 1528681 109750 403 544 8 1008554 89144 1174 1205

[0093] Example 6: Comparison of the effects of an oil-in-water composite adjuvant with fish oil as the oil phase and other adjuvants on the immune effect of recombinant novel coronavirus antigen protein

[0094] This example compares an oil-in-water composite adjuvant with fish oil as the oil phase with other adjuvants to further illustrate the immunological effect of the present invention. BALB / c mice of SPF grade at 6 to 8 weeks of age were divided into 10 mice per group. According to the animal experimental scheme shown in Table 8, the recombinant novel coronavirus antigen protein was mixed with different preparation formulas to prepare immune substances. Two intramuscular immunizations were performed at 0w and 2w, and blood was collected from the intracanthal vein at 3w to separate the serum. The serum was inactivated at 56°C for 30min, and the neutralizing antibody titer of the serum against the BF.7 novel coronavirus, and the mouse serum-specific IgG1 and IgG2a antibodies were detected using a live virus microneutralization test. The test results are as follows. Figure 8 and as shown in Table 9.

[0095] Table 8. Animal immunization schemes with different adjuvants

[0096]

[0097] Table 9. Results of mouse serum specific IgG antibody and neutralizing antibody detection

[0098] Group IgG1(GMT) IgG2a (GMT) Wild virus (GMT) 1 50 50 10 2 6553600 25600 2590 3 7023975 33779 3518 4 2483350 1426310 2445 5 1638400 400 608 6 1241675 1600 611 7 310419 400 259 8 6553600 409600 3707

[0099] Example 7: Stability of an oil-in-water composite adjuvant with fish oil as the oil phase

[0100] Stability testing was conducted using Formulation No. 3 (fish oil emulsion + Poly(I:C) (50 μg)) prepared in Example 6. After placement in constant temperature and humidity chambers at 4°C, 25°C, and 37°C, the formulation maintained a uniform milky white appearance, with no signs of delamination, flocculation, or precipitation. The particle size and polydispersity coefficient were measured after two weeks and one month, as shown in Table 10.

[0101] Table 10. Particle size and polydispersity coefficient at different times

[0102]

[0103] Comparative Example: Comparison of the effects of the mixture of Poly(I:C) in the second component of the present invention and different adjuvants on the immune effect of recombinant novel coronavirus antigen protein

[0104] This example compares the mixture of Poly (I: C) and different adjuvants with the second component of the present invention. BALB / c mice of SPF grade at 6 to 8 weeks of age were divided into 10 / group, and the recombinant novel coronavirus antigen protein was mixed with different formulations according to the animal experimental scheme shown in Table 11 to prepare immune substances. Three intramuscular immunizations were performed at 0w, 2w and 4w, and blood was collected from the intracanthal vein at 5w to separate the serum, which was inactivated at 56°C for 30min. The neutralizing antibody titer of the serum against the BA.1 novel coronavirus was detected by pseudovirus microneutralization test, and the mouse serum-specific IgG1 and IgG2a antibodies were detected by ELISA. The mouse spleen was taken at 6w, and the mouse spleen mononuclear cells were isolated. The mouse cellular immunity levels IFNγ, IL-5, and IL-17A were detected by ELISPOT experiment. The test results are as follows Figure 9 and as shown in Table 12.

[0105] Table 11. Animal immunization scheme of Poly(I:C) mixed with different adjuvants

[0106]

[0107]

[0108] Table 12. Results of mouse serum specific IgG antibodies, neutralizing antibodies and cytokines detection

[0109] Group IgG1(GMT) IgG2a (GMT) Pseudovirus (GMT) IFNγ IL-5 IL-17A 1 50 50 20 / / / 2 50 50 20 / / / 3 400 50 20 / / / 4 3057363 72408 5170 190 32 26 5 2661589 135118 6425 469 228 15 6 2317048 166349 4160 641 608 11 7 1882027 63035 4039 774 180 26 8 2317048 72408 2839 / / /

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A vaccine composition, characterized in that The invention comprises a first component and a second component, wherein the first component is a recombinant protein antigen, and the second component is an oil-in-water composite adjuvant, wherein the oil-in-water composite adjuvant comprises an aqueous phase, an oil phase, an emulsifier, and also comprises a Toll-like receptor agonist (TLR) and / or a stimulator of interferon genes (STING).

2. The vaccine composition according to claim 1, characterized in that The vaccine is an inactivated vaccine, a live attenuated vaccine, a genetically engineered vaccine, a subunit vaccine, a genetic reassortment vaccine, a DNA vaccine or an mRNA vaccine.

3. The vaccine composition according to claim 1, characterized in that The first component is a recombinant human papillomavirus protein antigen, a recombinant poliovirus protein antigen, a recombinant respiratory syncytial virus protein antigen or a recombinant novel coronavirus protein antigen; Preferably, the recombinant protein antigen is a recombinant novel coronavirus protein antigen.

4. The vaccine composition according to claim 1, characterized in that The oil-in-water composite adjuvant comprises 0.5 wt% to 1 wt% of an emulsifier, 5 wt% to 10 wt% of an oil phase, and further comprises a Toll-like receptor agonist with a final concentration of 0.5 to 5 mg / ml and / or a STING agonist with a final concentration of 100 to 300 μg / ml.

5. The vaccine composition according to claim 1, characterized in that The buffer of the aqueous phase includes one or more selected from citric acid / citrate buffer, phosphate buffer, carbonate buffer, acetate buffer, borate buffer, histidine buffer, tris(hydroxymethyl)methane buffer, succinate buffer or barbiturate buffer.

6. The buffer solution according to claim 5, characterized in that The buffer contains a final concentration of 0.01 to 1000 mM histidine and a final concentration of 0.01 to 1000 mM NaCl; the pH range of the buffer is 6.5 to 7.0; Preferably, the buffer is 10 mM histidine-150 mM NaCl, with a pH of 6.

8.

7. The vaccine composition according to claim 4, characterized in that The oil phase comprises one or more selected from soybean oil, peanut oil, fish oil, olive oil, sunflower oil, cottonseed oil, sesame oil, corn oil or coconut oil; Preferably, the oil phase is soybean oil and / or fish oil.

8. The vaccine composition according to claim 4, characterized in that The emulsifier comprises sorbitan fatty acid ester and / or a nonionic surfactant, wherein the sorbitan fatty acid ester is one or more selected from Span 20, Span 40, Span 60, Span 80 or Span 85, and the nonionic surfactant is one or more selected from Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Tween 81 or Tween 85; Preferably, the sorbitan fatty acid ester is Span 85, and the nonionic surfactant is Tween 80.

9. The vaccine composition according to claim 4, characterized in that The Toll-like receptor agonist is poly I-cytochrome P, and the STING agonist is cyclic dinucleotide.

10. The vaccine composition according to any one of claims 1 to 9, characterized in that The preparation method of the oil-in-water composite adjuvant comprises the following steps: Step 1) adding the Toll-like receptor agonist and / or STING agonist to a buffer solution to prepare solution I; Step 2) adding the oil phase and the emulsifier to the above solution I and stirring to obtain solution II; Step 3) High-pressure homogenization of the above solution II was performed at 1000-2000 bar for 6 times to prepare solution III; Step 4) The solution III is filtered and sterilized to finally prepare a solution IV, which is the oil-in-water composite adjuvant.

11. Use of the vaccine composition according to any one of claims 1 to 10 in the preparation of a medicament for treating or preventing viral infection.

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