Use of manganese tetroxide particles in the preparation of vaccine adjuvants

Abstract

The present invention discloses the use of trimanganese tetroxide particles in the preparation of a vaccine adjuvant. The adjuvant is a particle adjuvant, which is a trimanganese tetroxide particle that may or may not be externally encapsulated with an excipient, and the particle size of the particle adjuvant is 5 nm to 3000 nm. The trimanganese tetroxide particle adjuvant provided by the present invention can be effectively combined with a single-stranded nucleotide adjuvant, can effectively carry an immune antigen, and can achieve a better immunotherapy effect when using a smaller antigen dose and a relatively low injection amount, and can efficiently activate immune cells, achieving humoral balance and cellular immunity.

Description

[Technical field]

[0001] The present invention relates to the fields of biomedical and vaccine technology, and in particular to the use of trimanganese tetroxide particles in the preparation of vaccine adjuvants. [Background technology]

[0002] Vaccines refer to biological preparations made from various pathogenic microorganisms for use in vaccination. Since the development of vaccines, the control of many diseases, such as smallpox, has been achieved. To combat infectious diseases such as the new coronavirus, various vaccines, such as nucleic acid vaccines, inactivated virus vaccines, recombinant protein vaccines, viral vector vaccines, and subunit vaccines, have been developed. Among them, subunit vaccines are the most studied vaccines due to their safety, widespread use, and high degree of customizability. However, subunit vaccines are weakly immunogenic and therefore require repeated administration with adjuvants. Adjuvants, as non-specific immunostimulators, play an important role in inducing effective immune responses after vaccination.

[0003] Traditional aluminum adjuvants can induce effective humoral immune responses, but have difficulty in inducing cellular immune responses. There is increasing evidence that antibody and T cell immunity are required to effectively protect against the new coronavirus. However, aluminum adjuvants can only activate humoral immunity and lack the ability of mucosal immunity.

[0004] Manganese is an inorganic trace element nutrient required for a wide variety of physiological processes, such as development, reproduction, and neurological function. In recent years, the role of manganese as an immune stimulant has been gradually discovered. Besides manganese adjuvants can induce the production of type I interferon and cytokines in the absence of infection, manganese can also activate the cGAS-STING pathway to induce humoral and cellular immune responses. At present, there are reports on the application of divalent manganese and tetravalent manganese as vaccine adjuvants. One Chinese patent discloses the use of divalent manganese in the preparation of drugs for improving innate or / and adaptive immunity, manganese compositions containing divalent manganese for immune enhancement, and manganese dioxide nanoadjuvants and their preparation methods and uses. However, the immune enhancement effect of simple divalent manganese adjuvants or tetravalent manganese adjuvants needs to be improved.

[0005] CPG ODN (CpG oligonucleotide, or CpG oligodeoxynucleotide) is an artificially synthesized oligodeoxynucleotide (ODN) containing unmethylated cytosine guanine dinucleotide (CpG), which can mimic bacterial DNA and stimulate immune cells of various mammals, including humans. It enters via endocytosis, is recognized and bound by TLR9, activates the NF-κB pathway, produces a wide variety of cytokines, enhances antigen processing and presentation, and induces Th1 immune responses. A Chinese patent discloses the immune effect of a bivalent manganese adjuvant enhanced by CpG. When this adjuvant is used in a SARS-CoV-2 subunit vaccine, the required amounts of both Mn and CpG are relatively high (Emerg Microbes Infect 2021,10(1),1555-1573). Summary of the Invention

[0006] The present invention aims to overcome the above-mentioned shortcomings of the prior art and provide the use of trimanganese tetroxide particles in the preparation of vaccine adjuvants. The trimanganese tetroxide microparticle adjuvant provided by the present invention can be effectively combined with single-stranded nucleotide adjuvants and can effectively carry immune antigens to obtain adjuvant composition vaccines (FIG. 1), thereby achieving better immunotherapy effects with smaller antigen amounts and lower injection volumes, and can effectively deliver immune antigens to lymph node tissues to highly efficiently activate immune cells and achieve balanced humoral and cellular immunity. Compared with the prior art, the trimanganese tetroxide microparticle adjuvant of the present invention can achieve efficient targeted delivery effects and excellent immune activation effects using Mn element and CpG at a dose that is one-fifth that of the prior art and at a concentration that is one-fourth that of the prior art.

[0007] A first object of the present invention is to provide a use of trimanganese tetroxide particles in the preparation of a vaccine adjuvant.

[0008] A second object of the present invention is to provide an adjuvant composition.

[0009] A third object of the present invention is to provide a vaccine adjuvant.

[0010] A fourth object of the present invention is to provide a vaccine.

[0011] A fifth object of the present invention is to provide a method for preparing a vaccine.

[0012] In order to achieve the above object, the present invention is implemented by the following solutions.

[0013] There is provided a use of trimanganese tetroxide particles in the preparation of a vaccine adjuvant, the adjuvant being a particulate adjuvant consisting of trimanganese tetroxide particles which may or may not be externally encapsulated with an excipient, the particulate adjuvant having a particle size of 5 to 3000 nm.

[0014] Preferably, the trimanganese tetroxide particles are externally enveloped with an excipient, the excipient being one or more of a protein, a polypeptide, a polymer, a nucleic acid, and a polysaccharide, which can react with the single-stranded nucleotide adjuvant via non-covalent adsorption or chemically selective covalent modification, thereby binding the excipient to the single-stranded nucleotide adjuvant.

[0015] More preferably, the single stranded nucleotide adjuvant is an oligonucleotide, including a CpG ODN.

[0016] More preferably, the oligonucleotide is a DNA fragment, ATP, ADP, or AMP.

[0017] More preferably, the chemically selective covalent modification is based on pairs of chemically selective covalent modifying groups carried by the excipient and the single-stranded nucleotide adjuvant, including maleimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, succinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0018] More preferably, the polypeptides are extracted or chemically synthesized polypeptides consisting of different amino acid sequences.

[0019] More preferably, the polymer may be a polymer having amino or carboxyl groups on the surface, such as polyethylene glycol, polydopamine, or polyethylene glycol.

[0020] More preferably, the polysaccharide includes starch, glycogen, cellulose, chitin, inulin, agar, or hyaluronic acid.

[0021] More preferably, the molar ratio of elemental manganese to the excipient in the trimanganese tetroxide particles is (20-4000:1).

[0022] More preferably, the molar ratio of manganese element to excipient in the trimanganese tetroxide particles is (20-400:1).

[0023] Even more preferably, the molar ratio of elemental manganese to the excipient in the trimanganese tetroxide particles is (20-300:1).

[0024] Preferably, the trimanganese tetroxide particles are prepared as follows: a divalent manganese salt aqueous solution is thoroughly mixed with an excipient molecule solution to obtain a mixed solution, which is then mixed with an alkaline solution, thoroughly reacted, and purified by dialysis to obtain trimanganese tetroxide particles.

[0025] More preferably, the pH value of the solution is 6-14 after mixing with the alkaline solution.

[0026] More preferably, after mixing with the alkaline solution, the pH value of the solution is 6.5 to 7.4.

[0027] In the present invention, Mn 2+ , excipients and OH - By varying the molar ratio between

[0028] More preferably, the pH of the mixed solution is adjusted using one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, triethylamine, pyridine, N-methylmorpholine, and tetramethylethylenediamine.

[0029] More preferably, the concentration of the divalent manganese salt in the mixed solution is 0.01 to 0.5 mmol / mL, and the concentration of the excipient is 0.005 to 0.000125 mmol / mL.

[0030] More preferably, the conditions for sufficient mixing are as follows: the temperature is 30 to 37° C., and the system is reacted with stirring for 0.2 to 5 hours.

[0031] More preferably, the conditions for sufficient reaction are as follows: the system is reacted at a temperature of 30 to 37°C for 0.2 to 5 hours, and then heated to 60 to 95°C with stirring for 0.2 to 5 hours to promote crystal growth and stability.

[0032] An adjuvant composition is provided, the adjuvant complex comprising a particulate adjuvant and a single-stranded nucleotide adjuvant, the particulate adjuvant being comprised of trimanganese tetroxide particles which may or may not be externally encapsulated with an excipient.

[0033] Preferably, the single stranded nucleotide adjuvant is an oligonucleotide, including a CpG ODN.

[0034] More preferably, the oligonucleotide is a DNA fragment, ATP, ADP, or AMP.

[0035] More preferably, the DNA fragment is a modified or unmodified DNA fragment.

[0036] More preferably, the modified DNA fragments are DNA fragments that have been functionalized with an amino, carboxyl, or thiol functional group, or a chemically selective covalent modification group, including maleimidation, succinimidation, and 1-(3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride functionalization, so as to achieve specific binding of the modified DNA to the surface of the trimanganese tetroxide particles.

[0037] CpG ODN are classified into types A, B, and C according to the strong Th1-type response and cellular immune stimulatory activity that they induce. Type A CpG ODN induces plasmacytoid dendritic cells (pDCs) to produce large amounts of IFN-α and NF-κB, which are weak stimulators. Type B CpG ODN strongly activates B cells but weakly stimulates IFN-α secretion. Type C CpG ODN has both the characteristics of types A and B. Type C CpG ODN induces strong IFN-α production from pDCs and B cell stimulation.

[0038] In a specific embodiment, the single stranded nucleotide is tcgtcgttttcggcgcgcgccg-SH.

[0039] A vaccine adjuvant is provided, which is prepared by coupling a particulate adjuvant and a single-stranded nucleotide adjuvant in an adjuvant composition via chemically selective covalent modification in a buffer having a pH of 6-9 for 0.5-24 hours, and then purifying the reaction mixture.

[0040] The particulate adjuvant is composed of a trimanganese tetroxide particle externally encapsulated with an excipient, and the particulate adjuvant is bound to a single-stranded nucleotide adjuvant via covalent modification, and the particulate adjuvant is composed of a trimanganese tetroxide particle adjuvant that is not externally encapsulated with an excipient, and the adjuvant is bound to the single-stranded nucleotide adjuvant via adsorption.

[0041] Preferably, coupling by chemically selective covalent modification involves activating functional groups of the particulate adjuvant and the single-stranded nucleotide adjuvant in the adjuvant composition, and then coupling the particulate adjuvant and the single-stranded nucleotide adjuvant by chemically selective covalent modification using the activated functional groups.

[0042] More preferably, activating the functional groups of the particulate adjuvant comprises activating the amino or thiol groups with N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide and adjusting the pH to change the spatial structure of the excipient, and activating the functional groups of the single-stranded nucleotide comprises activating the carboxyl or phospholipid groups with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0043] Preferably, the particulate adjuvant and the single-stranded nucleotide are bound by EDC / NHS reaction, electrostatic adsorption or coordinate adsorption.

[0044] Preferably, the molar ratio of elemental manganese to the nucleotide adjuvant in the adjuvant composition is 1:(0.001-1000).

[0045] A vaccine is provided and includes an adjuvant composition and / or a vaccine adjuvant.

[0046] Preferably, the adjuvant composition and / or the vaccine adjuvant carries a vaccine antigen.

[0047] More preferably, the vaccine antigens include inactivated or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.

[0048] More preferably, the pathogen includes a virus, a bacterium and / or a parasite.

[0049] Even more preferably, the pathogen comprises a virus and / or a parasite.

[0050] Even more preferably, the virus is selected from a DNA virus or an RNA virus, in particular the virus is selected from the Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae families, more preferably the virus is selected from one or more of a novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

[0051] Even more preferably, the virus is selected from a novel coronavirus and / or an influenza virus.

[0052] Even more preferably, the bacteria is selected from gram-positive and gram-negative bacteria, in particular the bacteria is selected from one or more of Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, and Helicobacter pylori.

[0053] Even more preferably, the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.

[0054] A method for preparing a vaccine is provided, which comprises thoroughly mixing an adjuvant composition and / or a vaccine adjuvant with a vaccine antigen to obtain a vaccine.

[0055] Preferably, the method of thorough mixing involves repeatedly pumping the system with a syringe 50-200 times to mix uniformly, and then placing it on a rotary shaker and mixing for 10-60 minutes.

[0056] Preferably, the vaccine antigens include inactivated or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.

[0057] More preferably, the pathogen includes a virus, a bacterium and / or a parasite.

[0058] More preferably, the pathogen comprises a virus and / or a parasite.

[0059] Even more preferably, the virus is selected from a DNA virus or an RNA virus, in particular the virus is selected from the Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae families, more preferably the virus is selected from one or more of a novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

[0060] Even more preferably, the virus is selected from a novel coronavirus and / or an influenza virus.

[0061] Even more preferably, the bacteria is selected from gram-positive and gram-negative bacteria, in particular the bacteria is selected from one or more of Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, and Helicobacter pylori.

[0062] Preferably, the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.

[0063] Preferably, the method of administration of the adjuvant-vaccine conjugate reagent includes intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, mucosal administration and any combination thereof.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The trimanganese tetroxide particles provided by the present invention can be effectively combined with single-stranded nucleotide adjuvants, can effectively carry immune antigens, and can achieve better immunotherapeutic effects with smaller antigen doses and lower injection volumes. They can also efficiently activate immune cells to achieve balanced humoral and cellular immunity. [Brief description of the drawings]

[0066] [Figure 1]1 is a schematic diagram of the structure of an adjuvant composition vaccine, in which X1 can be a protein, polypeptide, polymer, nucleic acid, polysaccharide, or a polypeptide having a modifiable amino, carboxyl, or thiol group on its surface, and can also be a protein, polypeptide, polymer, nucleic acid, or polysaccharide containing Y1, Y1 is a chemically selective cross-linking group (maleimide, succinimide, etc.), ODN1 is a negatively charged single-stranded nucleotide (including CpG, CpG derivatives, and other single-stranded polynucleotides), ODN2 is a single-stranded nucleotide containing Y1 that can be bound to a manganese adjuvant containing X1, and ODN3 is a single-stranded nucleotide containing a functionalized amino, carboxyl, or thiol group that can be bound to a manganese adjuvant containing Y1. [Diagram 2] 1 shows TEM images of the trimanganese tetroxide microparticle adjuvants obtained in Examples 1 and 2 of the present invention. [Diagram 3] 1 is an XRD image of the trimanganese tetroxide microparticle adjuvant obtained in Example 1 of the present invention. [Figure 4] FIG. 1 shows the evaluation of BMDC activation and antigen presentation ability of the adjuvant composition vaccine constructed in Example 4 of the present invention and the vaccines of different experimental groups in Example 5. A is the result of the BMDC activation experiment, B is the result of MHC-I expression, and C is the result of MHC-II expression. [Diagram 5] 1 shows the results of antibody titers in mouse serum 19, 35, and 56 days after immunization with the adjuvant composition vaccine constructed in Example 4 of the present invention and the vaccines with different formulations in Example 5. [Figure 6] 1 shows the results of antibody levels related to cellular and humoral immunity in mouse serum 56 days after immunization with the adjuvant composition vaccine constructed in Example 4 of the present invention and the vaccines with different formulations in Example 5. [Figure 7] 1 shows the results of pseudovirus neutralizing antibodies in mouse serum 56 days after immunization with the adjuvant composition vaccine constructed in Example 4 of the present invention and the vaccines with different formulations in Example 5. [Figure 8]1 shows the results of specific antibody titers in mice two weeks after secondary immunization with MnCpG, Mn2CpG, or the commercial aluminum adjuvant Alum carrying influenza H1N1 HA antigen prepared in step A and step B of Example 4 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present invention will be described in detail below in conjunction with drawings and specific examples.The examples are only used to illustrate the present invention, and are not used to limit the scope of the present invention.Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used are, unless otherwise specified, reagents and materials available from commercial channels.

[0068] Primary Reagent Sources: SARS-CoV-2 RBD antigen was purchased from Sino Biological (cat. no. 40592-V08H4);

[0069] The experimental CpG adjuvant used in the exemplary embodiment of the present invention is Type C CpG-ODN 2395, [5'-tcgtcgttttcggcgcgcgccg-3'] (InvivoGen, product catalog number: tlrl-2395-1).

[0070] The thiolated CpG ODN (sequence: tcgtcgttttcggcgcgcgccg-SH) in the examples was custom synthesized by Sangon Biotech.

[0071] Carboxylic acid polyethylene glycol maleimide, COOH-PEG-MAL (manufacturer's catalog number PEG-MSCM), methoxypolyethylene glycol propionic acid, mPEG-COOH (manufacturer's catalog number mPEG-PA-2K), were purchased from Jinpan Biotech.

[0072] Commercially available aluminum adjuvant Alum (Invivogen, Alhydrogel® Adjuvant 2%, CAS: 21645-51-2)

[0073] Example 1 Preparation of Manganese Tetroxide Nanoparticle Adjuvant 240 ml of soluble manganese salt MnCl2·4H2O aqueous solution (0.25 mmol / ml) and 1 ml of excipient mixture solution of COOH-PEG-MAL (0.02 mmol / ml) and mPEG-COOH (0.18 mmol / ml) were thoroughly mixed to prepare Mn 2+ A premix solution with a molar ratio of 300:1 between 100 and total excipients was obtained.

[0074] The premixed solution was then added to the NaOH solution at a rate of 6.9 ml / min with a peristaltic pump while stirring at 700 rpm to mix and adjust the pH value of the solution to 6.9.

[0075] Finally, the mixture was reacted at 34°C for 30 minutes, and then heated to 80°C for 3 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0076] Example 2 Preparation of trimanganese tetroxide microparticle adjuvant 0.5 mmol / ml manganese salt MnCl2·4H2O was dissolved in 250 ml water, then added to 3 mmol / ml NaOH at a rate of 3.9 ml / min by peristaltic pump, mixed and reacted. After the reaction was completed, the pH value of the solution was adjusted to 6.9 by adding more 0.02 M NaOH.

[0077] The mixture was stirred at 500 rpm and reacted at 34° C. for 30 minutes, and finally heated to 60° C. for 3 hours. After the reaction was completed, the mixture was allowed to cool to room temperature, centrifuged, then washed with 0.5% sodium chloride, and finally sterilized by high-pressure steam or filtration membrane.

[0078] Example 3 Preparation of Manganese Tetroxide Nanoparticle Adjuvant 40 ml of manganese sulfate aqueous solution (0.1 mmol / mL) and 200 ml of ovalbumin excipient mixture solution were thoroughly mixed to obtain Mn 2+ A premix solution with a molar ratio of 200:1 between 100 and total excipients was obtained.

[0079] The premixed solution was then added to the KOH solution at a rate of 3.1 ml / min with a peristaltic pump while stirring at 1000 rpm to mix, and the pH value of the solution was adjusted to 7.4.

[0080] Finally, the mixture was reacted at 30° C. for 60 minutes, and then heated to 90° C. for 2 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0081] Example 4 Method for preparing trimanganese tetroxide nanoparticle adjuvant 200 ml of manganese nitrate aqueous solution (0.2 mmol / mL) and 100 ml of the cyclic RGD polypeptide excipient mixture solution were thoroughly mixed to obtain Mn 2+ A premix solution with a molar ratio of 2:1 between 100 and total excipients was obtained.

[0082] The premixed solution was then added to the Ca(OH)2 solution at a rate of 5.8 ml / min with a peristaltic pump while stirring at 1400 rpm to mix, and the pH value of the solution was adjusted to 6.5.

[0083] Finally, the mixture was reacted at 20° C. for 90 minutes, and then heated to 70° C. for 5 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0084] Example 5 Preparation of Manganese Tetroxide Nanoparticle Adjuvant 150 ml of manganese acetate solution (0.6 mmol / mL) and 300 ml of starch (5000 Da) excipient mixture were thoroughly mixed to obtain Mn 2+ A premix solution with a molar ratio of 100:1 between 1 and the total excipients was obtained.

[0085] The premixed solution was then added to the aqueous ammonia solution at a rate of 7.2 ml / min with a peristaltic pump while stirring at 900 rpm to mix, and the pH value of the solution was adjusted to 7.1.

[0086] Finally, the mixture was reacted at 35°C for 40 minutes, and then heated to 80°C for 2 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0087] Example 6 Method for preparing trimanganese tetroxide nanoparticle adjuvant 50 ml of manganese chloride aqueous solution (0.1 mmol / mL) and 200 ml of the aptamer-excipient mixed solution were mixed thoroughly to obtain Mn 2+ A premix solution with a molar ratio of 5:1 between 100 and total excipients was obtained.

[0088] The premixed solution was then added to the triethylamine solution at a rate of 1.8 ml / min with a peristaltic pump while stirring at 1400 rpm to mix, and the pH value of the solution was adjusted to 7.0.

[0089] Finally, the mixture was reacted at 15° C. for 90 minutes, and then heated to 60° C. for 5 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0090] Example 7 Method for preparing trimanganese tetroxide nanoparticle adjuvant 350 ml of manganese chloride aqueous solution (0.3 mmol / mL) and 200 ml of polyethyleneimine excipient mixed solution were thoroughly mixed to obtain Mn2+ A premix solution with a molar ratio of 50:1 between 100 and total excipients was obtained.

[0091] The premixed solution was then added to the triethylamine solution at a rate of 4.8 ml / min with a peristaltic pump while stirring at 900 rpm to mix, and the pH value of the solution was adjusted to 7.1.

[0092] Finally, the mixture was reacted at 35° C. for 40 minutes, and then heated to 90° C. for 2 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0093] Example 8 Method for preparing trimanganese tetroxide nanoparticle adjuvant 450 ml of manganese chloride aqueous solution (0.2 mmol / mL) and 300 ml of chitin excipient mixed solution were thoroughly mixed to obtain Mn 2+ A premix solution with a molar ratio of 250:1 between 100 and total excipients was obtained.

[0094] The premixed solution was then added to the triethylamine solution with a peristaltic pump at a rate of 6.9 ml / min while stirring at 1100 rpm to mix, and the pH value of the solution was adjusted to 6.7.

[0095] Finally, the mixture was reacted at 25°C for 80 minutes, and then heated to 80°C for 2 hours to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0096] Example 9 Method for preparing trimanganese tetroxide nanoparticle adjuvant 250 ml of manganese chloride aqueous solution (0.5 mmol / mL) and 200 ml of glycogen excipient mixed solution were mixed thoroughly to obtain Mn 2+ A premix solution with a molar ratio of 50:1 between 100 and total excipients was obtained.

[0097] The premixed solution was then added to the triethylamine solution at a rate of 3.8 ml / min with a peristaltic pump while stirring at 1300 rpm to mix, and the pH value of the solution was adjusted to 7.2.

[0098] Finally, the mixture was reacted at 35° C. for 40 minutes, and then heated to 90° C. for 1 hour to promote the growth and stability of the crystalline morphology of the particles. After the reaction was completed, the mixture was left to cool to room temperature, then centrifuged, washed, and finally sterilized by high pressure steam or filtration membrane for later use.

[0099] Example 10. Characterization of the Physicochemical Properties of Manganous Trioxide Microparticle Adjuvants I. Observation under an electron microscope 1. Experimental Method (1) At 25° C., the trimanganese tetroxide microparticle adjuvants prepared in each of Examples 1 and 2 were diluted to a concentration of 10 μg / ml and dropped onto a common carbon support film. The structure of the trimanganese tetroxide microparticle adjuvant was observed under an electron microscope (FEI Company, model: Tecnai G2 20S-TWIN).

[0100] 2. Experimental Results The results are shown in FIG. 2, which shows TEM images of the trimanganese tetroxide particles prepared in Example 1 (left) and Example 2 (right).

[0101] As shown in Figure 2, the trimanganese tetroxide particulate adjuvants prepared in Examples 1 and 2 are both particulate. When excipients were present, the average size of the resulting manganese particulate adjuvants was 13.1 nanometers, whereas in the absence of excipients, the resulting product consisted of micron-sized particles formed by aggregation of smaller particles, with an average particle size of 1582.1 nm, distributed in the range of 350-3000 nm.

[0102] II. XRD characterization of manganous oxide microparticle adjuvants 1. Experimental Method 10 ml of the trimanganese tetroxide microparticle adjuvant prepared in Example 1 in the presence of excipients was freeze-dried to obtain a powder sample which was characterized by XRD and then analyzed by Jade.

[0103] 2. Experimental Results The results are shown in FIG. 3 and indicate that the crystal morphology of the manganese particulate adjuvant obtained in Example 1 corresponds to Mn3O4.

[0104] III. Characterization of particle size and surface charge of manganese tetroxide particulate adjuvants 1. Experimental Method At 25°C, the concentration of the trimanganese tetroxide microparticle adjuvants prepared in Examples 1 to 9 of the present invention was diluted to 10 μg / ml, and the hydrated particle size of the trimanganese tetroxide microparticle adjuvants was tested using a nanoparticle size analyzer (purchased from Malvern, Zetasize analyzer Nano ZS model). The results are shown in Table 1.

[0105] As shown by the TEM results, when the particles prepared in Example 2 without the presence of excipients are compared to the trimanganese tetroxide particles prepared in Examples 1 and 3-9 with the presence of excipients, the presence of the excipients results in particles that are smaller in size, more uniform in distribution, better dispersible, and more stable in aqueous solution. [Table 1]

[0106] Example 11 Preparation of SARS-CoV-2 RBD Recombinant Protein Adjuvant Composition Vaccine Combined with Manganese Tetroxide Nanoparticle Adjuvant and CpG A) 5 ml of the aqueous solution (0.125 mmol / ml) of the manganese tetroxide particles prepared in Example 1 was mixed with an equal volume of thiol-modified CpG (0.0004 mmol / ml) (purchased from Sangon Biotech, tcgtcgttttcggcgcgcgccg-SH). The mixture was reacted at room temperature with stirring at 300 rpm for 2 hours while maintaining the pH at 8.5, and purified by ultrafiltration to complete the preparation of the adjuvant composition of manganese tetroxide nanoparticle adjuvant and CpG, and the adjuvant composition of manganese tetroxide nanoparticle adjuvant and CpG (adjuvant composition MnCpG) was obtained.

[0107] B) 5ml of the aqueous solution (0.125mmol / ml) of the manganese tetroxide particles prepared in Example 2 was mixed with an equal volume of C-type CpG ODN2395 (0.0004mmol / ml) (5'-tcgtcgttttcggcgcgcgccg-3'). The mixture was reacted at room temperature with stirring at 300 rpm for 2 hours while maintaining the pH at 8.5, and purified by ultrafiltration to complete the preparation of the adjuvant composition of manganese tetroxide nanoparticle adjuvant and CpG-ODN2395, and the adjuvant composition of manganese tetroxide nanoparticle adjuvant and CpG (adjuvant composition Mn2CpG) was obtained.

[0108] C) The adjuvant compositions prepared in steps A and B of this example were dispersed in saline, and 10 μg of SARS-CoV-2 recombinant RBD antigen (purchased from Sino Biological, catalog number 40592-V08H4) was added to each of them. The system was pumped 100 times with a syringe. After uniform mixing, the mixture was placed on a rotary shaker and mixed for 10 to 60 minutes to obtain an adjuvant composition vaccine carrying an antigen (SARS-CoV-2 RBD recombinant subunit) based on the combination of manganese tetroxide nanoparticle adjuvant and CpG adjuvant.

[0109] Example 12 Evaluation of BMDC activation and antigen presentation ability of adjuvant composition vaccine I. Experimental Method 1. Experimental Grouping (1) Ctr (control group): The injection volume was 100 μL, and the injection solution was saline; (2) Ag group: the injection volume was 100 μL, and 10 μg of RBD antigen was injected; (3) CpG-Ag group: the injection volume was 100 μL, and 10 μg of RBD antigen and CpG-ODN2395 (purchased from InvivoGen, product catalog number: tlrl-2395-1) composite adjuvant was injected; (4) Mn-Ag group: 25 μg of the manganese-containing nanoparticle adjuvant prepared in Example 1 of the present invention was injected, carrying 10 μg of RBD antigen (Ag) in an injection volume of 100 μL; (5) Alum-Ag group: injected with 50 μg of commercial aluminum adjuvant Alum (Invivogen, Alhydrogel® adjuvant 2%, CAS: 21645-51-2), carrying 10 μg of RBD antigen, with an injection volume of 100 μL; (6) MnCpG-Ag group: The adjuvant composition vaccine constructed in step A of Example 4, i.e., an adjuvant composition of manganese tetroxide nanoparticle adjuvant and CpG (adjuvant composition MnCpG) was injected in an injection volume of 100 μl carrying 10 μg of RBD antigen (Ag).

[0110] 2. Experimental Method BMDC cells were plated at 3 x 10 per well in a 6-well plate. 5 BMDCs were seeded with cells and grown overnight. Then, the materials of the six different experimental groups were added respectively, and after further incubation for 24 hours, the cells were harvested and stained with anti-CD11c, anti-CD80 and anti-CD86, anti-MHC-1, and anti-MHC-II flow staining solution. The expression levels of costimulatory factors CD80, CD86, and antigen recognition signals MHC-1 and MHC-II on the surface of BMDCs were measured by flow cytometer.

[0111] II. Experimental Results The results are shown in Figure 4. Figure 4 shows the evaluation of the BMDC activation and antigen presentation ability of a vaccine prepared using the adjuvant composition MnCpG constructed in Example 11 of the present invention and other vaccines with different formulations in Example 6. Figure 4A shows the results of a BMDC activation experiment, Figure 4B shows the results of MHC-I expression, and Figure 4C shows the results of MHC-II expression.

[0112] As shown in Figure 4, the adjuvant composition vaccine constructed in Example 11 has a better BMDC activation effect and higher antigen presentation ability than the single adjuvant vaccine. The adjuvant composition (MnCpG group) prepared in Example 1 of the present invention can stimulate the activation of BMDC by about 30.4, which is 2.28 times that of the simple CpG group. In terms of antigen presentation ability, the MnCpG-Ag group also shows a better antigen presentation effect than the simple CpG group.

[0113] Example 13 Evaluation of the efficacy of adjuvant composition vaccines in mouse immunization I. Experimental Method 1. Experimental Grouping (1) Ctr group (control group): The injection volume was 100 μL, and the injection solution was saline; (2) Ag group: the injection volume was 100 μL, and 10 μg of RBD antigen was injected; (3) CpG-Ag group: the injection volume was 100 μL, and 10 μg of RBD antigen and CpG-ODN2395 (purchased from InvivoGen, product catalog number: tlrl-2395-1) adjuvant complex was injected; (4) Mn-Ag group: injected with 25 μg of the nanovaccine constructed by the manganese-containing microparticle adjuvant prepared in Example 1 of the present invention carrying 10 μg of RBD antigen (Ag), with an injection volume of 100 μL; (5) Alum-Ag group: the injection volume was 100 μL, and 50 μg of the commercial aluminum adjuvant Alum (Invivogen, Alhydrogel® adjuvant 2%, CAS: 21645-51-2) was injected, carrying 10 μg of RBD antigen; (6) MnCpG-Ag group: The adjuvant composition vaccine constructed in step A of Example 11, i.e., an adjuvant composition of manganese tetroxide nanoparticles and CpG (adjuvant composition MnCpG), was injected in an injection volume of 100 μl carrying 10 μg of RBD antigen (Ag). (7) Mn2CpG-Ag group: The vaccine constructed in step B of Example 11, i.e., an adjuvant composition of manganese tetroxide microparticles and CpG (adjuvant composition Mn2CpG), carrying 10 μg of RBD antigen (Ag) in an injection volume of 100 μl, was injected.

[0114] 2. Immunization of Mice Under the premise of following the national animal health protocol, 6-8 week old BALB / c mice were selected for two doses of vaccination. The six vaccines in the above categories were intramuscularly injected at 100μl / mouse for immunization of mice, with 5 mice per group, for a total of 7 groups. The first vaccination date was day 0, the second vaccination date was day 21, and serum samples were collected on days 19, 35, and 56.

[0115] B) Detection of IgG titers in mouse serum The IgG titer levels in mouse serum induced by the vaccines in the different experimental groups in step A were evaluated by conventional enzyme-linked immunosorbent assay (ELISA).

[0116] Mouse serum was serially diluted proportionally, and the diluted serum was added to a 96-well ELISA plate precoated with RBD antigen (2 μg / ml) and incubated for 2 hours at 37°C. After washing the plate, diluted HRP-conjugated goat anti-mouse IgG antibody (dilution 1:2000) was added at 100 μl per well. The plate was incubated for 1.5 hours at 37°C, washed, and then incubated with TMB colorimetric solution. The reaction was stopped by adding stop buffer, and the absorbance at OD450 was read using a microplate reader.

[0117] C) Detection of antibody titers biased towards cellular and humoral immunity With reference to the above-mentioned method for testing IgG titers, sera were collected from mice 56 days after immunization in step A of Example 6 of the present invention, and the levels of antibodies biased towards cellular and humoral immunity induced by the vaccine were evaluated.

[0118] II. Experimental Results The specific IgG antibody titers of the groups are shown in Figure 5. Figure 5 shows the RBD-specific IgG antibody titers in mouse serum 19 days, 35 days, and 56 days after immunization with the adjuvant composition vaccines constructed in Example 4 (MnCpG-Ag group and Mn2CpG-Ag group) and the other groups in this example.

[0119] As can be seen from Figure 5, the adjuvant composition vaccine (MnCpG-Ag group and Mn2CpG-Ag group) constructed in Example 11 can effectively enhance the antibody response induced by the SARS-CoV-2 vaccine in mice. The detection of serum antibodies at several dilutions shows that the adjuvant composition vaccine (MnCpG-Ag group and Mn2CpG-Ag group) constructed in Example 11 can effectively enhance the IgG immune response level. When carrying the same antigen dose (10 micrograms each), the antibody titer levels of the adjuvant composition vaccine groups constructed in Example 8 (MnCpG-Ag group and Mn2CpG-Ag group) were four times the antibody titer level of the Mn-Ag group vaccine using a simple manganese tetroxide microparticle adjuvant, 10 times the antibody titer level of the simple Alum adjuvant vaccine, and 50 times the antibody titer level of the simple CpG adjuvant group vaccine, indicating that the complex adjuvant with CpG can enhance the immune-enhancing ability of the manganese tetroxide microparticle adjuvant.

[0120] The antibody results on day 56 showed that the antibodies produced by the MnCpG-Ag group of Example 12 were 2.2 times higher than those of the Mn2CpG group, indicating that the nanoparticle adjuvant bound to CpG by covalent modification can generate higher antibody titers than the particulate vaccine bound to CpG by adsorption. Furthermore, the antibody titer of the Mn2CpG-Ag group was twice that of the Mn-Ag group, indicating that the antibodies produced were higher than those of the group using the simple trimanganese tetroxide adjuvant, even when the adjuvant composition was prepared by adsorption, indicating that the binding of CpG to the trimanganese tetroxide particulate adjuvant, whether via adsorption (Example 2) or covalent modification (Example 1), can significantly enhance the synergistic ability of their respective vaccines compared to the simple antigen-Ag group and the CpG-Ag group, promoting high antibody production levels.

[0121] A comparison of the balance between cellular immunity and humoral immunity induced by the adjuvant composition vaccine constructed in Example 11 is shown in Figure 6. Figure 6 shows the balance evaluation of cellular immunity and humoral immunity induced by the adjuvant composition vaccine constructed in Example 11 of the present invention and the vaccines with different formulations in Example 12. As shown in Figure 6, the IgG2C / IgG1 ratio induced by the vaccines prepared using the adjuvant compositions MnCpG and Mn2CpG obtained in Example 11 was close to 1, inducing a balanced T cell response, whereas the IgG2C / IgG1 ratio of the Mn-Ag group was less than 1, indicating that the simple manganese adjuvant Mn-Ag group induced an immune response biased toward Th2, and the CpG adjuvant vaccine induced an immune response biased toward Th1.

[0122] Example 14 Neutralizing antibody responses induced by adjuvant composition vaccines I. Experimental Method Serum samples from mice in each group 56 days after immunization with the vaccine obtained in step A of Example 12 were subjected to a pseudovirus infection neutralization test. The specific steps were as follows: the supernatant containing the pseudovirus (50 μl; purchased from Sino Biological, catalog number: PSV001) was pre-incubated with serially diluted mouse serum at 37°C for 1 hour, and then incubated with ACE2-expressing 293T cells (5 × 10 4 Fresh medium was added after 24 h and cells were lysed using a commercially available cell lysis buffer. Relative luciferase activity was measured in a luminometer (Bio-Tech) after addition of luciferase substrate. Pseudovirus neutralization efficiency was calculated and expressed as 50% neutralizing antibody titer.

[0123] II. Experimental Results The results are shown in Figure 7. Figure 7 shows a comparison of the in vitro immune effects against the pseudovirus of the adjuvant composition vaccine constructed in Example 11 and the vaccine of another formulation constructed in Example 12. As shown in Figure 7, the vaccine prepared using MnCpG and Mn2CpG of the adjuvant composition obtained in Example 11 can induce a significant enhancement of the neutralizing antibody response against the new coronavirus. However, the sera of the MnCpG-Ag group and the Mn2CpG-Ag group against the new coronavirus pseudovirus have the same neutralizing ability.

[0124] Example 15 Evaluation of adjuvant compositions for enhancing the protective effect of influenza subunit vaccines I. Preparation of Influenza Subunit Vaccines with Adjuvant Compositions The adjuvant composition MnCpG and the adjuvant composition Mn2CpG prepared in step A or B of Example 11 were dispersed in saline to prepare a 1 mg / ml solution, and 100 microliters of the solution was taken with a pipette, and 5 μg of influenza subunit antigen (referred to as HlNl HA, purchased from Sino Biological, catalog number 40731-V07H) was added. The system was mixed well by shaking or stirring, and then placed on a rotary shaker for 10 to 60 minutes to obtain an influenza subunit vaccine based on the adjuvant composition of manganese tetroxide microparticle adjuvant and CpG adjuvant, carrying influenza subunit antigens MnCpG-HA and Mn2CpG-HA.

[0125] II. Evaluation of antibody titers in mice immunized with influenza subunit vaccines The prepared adjuvant compositions, influenza subunit vaccines MnCpG-HA and Mn2CpG-HA, were administered intramuscularly for immunization, and the produced antigen-specific IgG and IgM antibody titers were measured.

[0126] The specific steps are as follows: Experimental animals: Balb / C mice, 6-8 weeks old, 5 mice / group, female.

[0127] Dosage: HlNl HA antigen 5 μg / mouse; commercial aluminum adjuvant Alum (purchased from Invivogen, CAS: 21645-51-2): 100 μg / 100 μl / mouse; MnCpG-HA and Mn2CpG-HA: 100 μg / 100 μl / mouse;

[0128] Experimental groups and doses: (1) HA group: HlNl HA antigen 5 μg / mouse; (2) Alum-HA group: commercial aluminum adjuvant Alum 100 μg / 100 μl + H1N1 HA 5 μg / mouse; (3) MnCpG-HA group: 100 μg MnCpG and 100 μl injection of HlN1 HA antigen 5 μg / mouse; (4) Mn2CpG-HA group: 100 μg Mn2CpG and 100 μl injection of HlNl HA antigen 5 μg / mouse.

[0129] Immunization regimen: Mice were immunized with the vaccine and then boosted according to the immunization group 3 weeks after the first immunization. Two weeks after the second immunization, blood was collected from the orbit and the antibody titer in the serum was measured.

[0130] Serum antibody titers were detected by enzyme-linked immunosorbent assay (ELISA) to assess vaccine-induced IgG and IgM levels in mouse serum.

[0131] III. Experimental Results FIG. 8 shows the results of an analysis of the titers of specific antibodies produced after immunization of mice with adjuvant compositions MnCpG and Mn2CpG prepared in step A and step B of Example 11 of the present application, carrying influenza H1N1 HA antigen, or with the commercially available aluminum adjuvant Alum.

[0132] The experimental results are shown in Figure 8A. The vaccines prepared using the adjuvant composition MnCpG and the adjuvant composition Mn2CpG obtained in step A and step B of Example 11 of the present invention enhance immune responses better than the vaccines prepared using commercially available aluminum adjuvants, and the antibody titers generated are 137 times and 96 times higher than those generated by the commercially available aluminum adjuvant, respectively.

[0133] The experimental results are shown in Figure 8B. The vaccines prepared using the adjuvant composition MnCpG and the adjuvant composition Mn2CpG obtained in step A and step B of Example 11 of the present invention have higher IgM levels than the vaccines prepared using the commercially available aluminum adjuvant, and the antibody titers generated are 4.3-fold and 3.1-fold higher than those generated by the aluminum adjuvant, respectively.

[0134] The results in FIG. 8 show that the vaccines prepared using the adjuvant compositions MnCpG and Mn2CpG prepared in Steps A and B of Example 11 have more effective immune enhancing capabilities than the vaccines prepared using commercially available aluminum adjuvants.

[0135] Example 16 Preparation of an adjuvant composition comprising a manganese tetroxide nanoparticle adjuvant combined with other oligonucleotides A) 5 ml of MES buffer solution (about pH 6) in which the solution of trimanganese tetroxide particles prepared in Example 3 (5 mg / ml) was dissolved was mixed with 1 ml of MES buffer solution (about pH 6) in which EDC solution (0.02 mmol / ml) was dissolved, and the mixture was activated at 35° C. for 30 minutes. Then, 1 ml of MES buffer solution (about pH 6) in which NHS solution (0.02 mmol / ml) was dissolved was added, and activation was continued for 2 hours. Then, unreacted EDC and NHS as well as reaction by-products were removed by dialysis, and then 2 ml of ATP solution (0.04 mmol / ml) was added, and the system was mixed. The pH was maintained at 8.0, and the mixture was reacted at room temperature with stirring at 300 rpm for 2 hours, and purified by ultrafiltration to complete the preparation of the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP, thereby obtaining the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP (adjuvant composition Mn3ATP).

[0136] B) 10 ml of MES buffer solution (about pH 5.7) in which the solution of trimanganese tetroxide particles prepared in Example 4 (10 mg / ml) was dissolved was mixed with 2 ml of MES buffer solution (about pH 5.7) in which EDC solution (0.05 mmol / ml) was dissolved, and the mixture was activated at 35° C. for 30 minutes. Then, 2 ml of MES buffer solution (about pH 5.7) in which NHS solution (0.05 mmol / ml) was dissolved was added, and activation was continued for 2 hours. Then, unreacted EDC and NHS as well as reaction by-products were removed by dialysis, and then 5 ml of ADP solution (0.1 mmol / ml) was added, and the system was mixed. The pH was maintained at 7.8, and the mixture was reacted at room temperature with stirring at 600 rpm for 15 hours, and purified by ultrafiltration to complete the preparation of the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP, thereby obtaining the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP (adjuvant composition Mn4ADP).

[0137] C) 5 ml of MES buffer solution (about pH 5.7) in which the manganese tetroxide particles prepared in Example 5 were dissolved (10 mg / ml) was mixed with 2 ml of EDC solution (0.05 mmol / ml) in MES buffer solution (about pH 5.7) and activated at 35° C. for 30 minutes. Then, 2 ml of maleimide solution (0.05 mmol / ml) in MES buffer solution (about pH 8.7) was added, and activation was continued for 2 hours. Then, unreacted EDC and NHS as well as reaction by-products were removed by dialysis, and then 5 ml of AMP solution (0.1 mmol / ml) was added and the system was mixed. The pH was maintained at 8.7, and the mixture was reacted at room temperature with stirring at 900 rpm for 24 hours, and purified by ultrafiltration to complete the preparation of the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP, thereby obtaining the adjuvant composition of manganese tetroxide nanoparticle adjuvant and ATP (adjuvant composition Mn5AMP).

[0138] D) 5 ml of the aqueous solution (0.2 mmol / ml) of the manganese tetroxide particles prepared in Example 6 was mixed with an equal volume of thiol-modified CpG (0.0005 mmol / ml) (purchased from Sangon Biotech, tcgtcgttttcggcgcgcgccg-SH). The mixture was reacted at room temperature with stirring at 500 rpm for 2 hours while maintaining the pH at 8.4, and purified by ultrafiltration to complete the preparation of the manganese tetroxide nanoparticle adjuvant and CpG adjuvant composition, and the manganese tetroxide nanoparticle adjuvant and CpG adjuvant composition (adjuvant composition Mn6CpG) was obtained.

[0139] Example 17 Preparation of a conjugate vaccine and evaluation of enhanced protective effect against parasites According to toxoplasmosis-related defense studies, the novel dense granulin (GRA) is one of the main components of excretory-secretory antigens (ESAs) secreted by the parasite during active invasion into host cells and is associated with the intracellular maintenance of the parasite. Related studies have shown that it can be used as a vaccine drug.

[0140] Experimental mice were randomly divided into groups with 10 mice in each group. (1) Ctr group (control group): The injection volume was 100 μL, and the injection solution was saline; (2) Ag group: the injection volume was 100 μL, and 10 μg of GRA antigen was injected; (3) ATP-Ag group: the injection volume was 100 μL, and 10 μg of GRA antigen bound to ATP adjuvant was injected; (4) ADP-Ag group: the injection volume was 100 μL, and 10 μg of GRA antigen bound to ADP adjuvant was injected; (5) Mn3-Ag group: the injection volume was 100 μL, and 25 μg of the nanovaccine constructed by the manganese-containing microparticle adjuvant prepared in Example 3 of the present invention carrying 10 μg of GRA antigen was injected; (6) Mn4-Ag group: injected with 50 μg of commercial aluminum adjuvant Alum (Invivogen, Alhydrogel® adjuvant 2%, CAS: 21645-51-2), carrying 10 μg of RBD antigen, with an injection volume of 100 μL; (7) Mn3ATP-Ag group: The adjuvant composition vaccine constructed in step A of Example 16, i.e., Mn3ATP, carrying 10 μg of RBD antigen (Ag) in an injection volume of 100 μl, was injected. (8) Mn4ADP-Ag group: the vaccine constructed in step B of Example 16, i.e., Mn4ADP, carrying 10 μg of RBD antigen (Ag) in an injection volume of 100 μl was injected.

[0141] The adjuvant compositions prepared in Example 16 were mixed with 10 μg of GRA vaccine drug, and the group without adjuvant composition was used for comparison, and experimental mice were immunized by injection. Five weeks after injection, the mice in each experimental group were infected with Toxoplasma gondii RH strain. The survival rate of the mice in each experimental group was observed and recorded daily. The experimental results are shown in the following table. [Table 2]

[0142] From this table, it can be seen that the trimanganese tetroxide nanoadjuvants constructed in Examples 3 and 4, and the composite nanoadjuvants Mn3ATP and Mn4ADP constructed in Example 16 can significantly enhance the immune effect of the original GRA vaccine.

[0143] Finally, it should be noted that the above examples are not intended to limit the scope of protection of the present invention, but are used only to describe the technical solutions of the present invention. Those skilled in the art can make other different forms of changes or modifications based on the above descriptions and ideas. It is unnecessary and impossible to list all the embodiments here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. 1. A use of trimanganese tetroxide particles in the preparation of a vaccine adjuvant, the adjuvant being a particulate adjuvant consisting of trimanganese tetroxide particles which may or may not be externally encapsulated with an excipient, the particulate adjuvant having a particle size of 5 to 3000 nm.

2. 2. The use of claim 1, wherein the trimanganese tetroxide particles are externally enveloped with an excipient which is one or more of a protein, a polypeptide, a polymer, a nucleic acid, and a polysaccharide, and the excipient is capable of reacting with a single-stranded nucleotide adjuvant via chemically selective covalent modification.

3. The use according to claim 2, wherein the mononucleotide adjuvant is an oligonucleotide comprising a CpG ODN.

4. 3. The use of claim 2, wherein the chemically selective covalent modification is based on a pair of chemically selective covalent modifying groups carried by the excipient and the single-stranded nucleotide adjuvant, and the chemically selective covalent modification includes maleimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, succinimide and / or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

5. The use according to claim 2, wherein the molar ratio of manganese element in the trimanganese tetroxide particles to the excipient is (20-4000):

1.

6. The use according to claim 5, wherein the molar ratio of manganese element in the trimanganese tetroxide particles to the excipient is (20-400):

1.

7. The use according to claim 6, wherein the molar ratio of manganese element in the trimanganese tetroxide particles to the excipient is (20-300):

1.

8. The use according to any one of claims 2 to 7, in which the trimanganese tetroxide particles externally wrapped with the excipient are prepared, i.e., a divalent manganese salt aqueous solution and an excipient molecule solution are thoroughly mixed to obtain a mixed solution, which is then mixed with an alkaline solution to thoroughly react with each other, purified by dialysis, and freeze-dried to obtain the trimanganese tetroxide particles externally wrapped with the excipient.

9. The use according to claim 8, wherein after mixing with the alkaline solution, the pH value of the solution is between 6 and 14.

10. The use according to claim 9, wherein after mixing with the alkaline solution, the pH value of the solution is between 6.5 and 7.

4.

11. The use according to claim 8, wherein the conditions for sufficient mixing are a temperature of 30-37°C and reacting the system with stirring for 0.2-5 hours.

12. 9. The use according to claim 8, wherein the conditions for sufficient reaction are first reacting the system at a temperature of 30-37° C. for 0.2-5 hours, and then heating the system to a temperature of 60-95° C. for 0.2-5 hours.

13. An adjuvant composition comprising a microparticle adjuvant and a single-stranded nucleotide adjuvant, the microparticle adjuvant being either externally encapsulated with an excipient or not, the adjuvant composition being the trimanganese tetroxide particle described in any one of claims 1 to 7.

14. The adjuvant composition of claim 13, wherein the single-stranded nucleotide adjuvant is an oligonucleotide, including a CpG ODN.

15. The adjuvant composition of claim 14, wherein the oligonucleotide is a DNA fragment, ATP, ADP and / or AMP.

16. The adjuvant composition of claim 15, wherein the DNA fragment is a modified or unmodified DNA fragment.

17. 17. The adjuvant composition of claim 16, wherein the modified DNA fragments are DNA fragments functionalized with amino, carboxyl or thiol functional groups, or chemically selective covalent modification groups, including maleimidation, succinimidation, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride functionalization modifications.

18. 18. The adjuvant composition of claim 17, wherein the single stranded nucleotide adjuvant is tcgtcgttttcggcgcgcgccg-SH.

19. 16. A vaccine adjuvant, prepared by coupling the particulate adjuvant in the adjuvant composition of claim 15 with the single-stranded nucleotide adjuvant via non-covalent adsorption or chemically selective covalent modification in a buffer solution of pH 6-9 for 0.5-24 hours, and then purifying the reaction mixture.

20. 20. The vaccine adjuvant of claim 19, wherein the particulate adjuvant is a manganese tetroxide particle externally encapsulated with an excipient, and the particulate adjuvant is bound to the single-stranded nucleotide adjuvant via covalent modification, and the particulate adjuvant is a manganese tetroxide particle not externally encapsulated with an excipient, and the particulate adjuvant is bound to the single-stranded nucleotide adjuvant via adsorption.

21. 20. The vaccine adjuvant of claim 19, wherein the coupling by the chemically selective covalent modification reaction involves activating a functional group of the particulate adjuvant and a functional group of the single-stranded nucleotide adjuvant in the adjuvant composition, and then coupling the particulate adjuvant and the single-stranded nucleotide adjuvant by the chemically selective covalent modification reaction using the activated functional group.

22. 20. The vaccine adjuvant of claim 19, wherein the particulate adjuvant and the single-stranded nucleotide are bound by EDC / NHS reaction, electrostatic adsorption, or coordinate adsorption.

23. 20. A vaccine comprising the adjuvant composition of claim 13 and / or the vaccine adjuvant of claim 19.

24. 24. The vaccine of claim 23, wherein the adjuvant composition of claim 13 and / or the vaccine adjuvant of claim 19 carries a vaccine antigen.

25. 25. The vaccine of claim 24, wherein the vaccine antigens include inactivated or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.

26. 26. The vaccine of claim 25, wherein the pathogen comprises a virus, a bacterium and / or a parasite.

27. 27. The vaccine of claim 26, wherein the pathogen comprises a virus and / or a parasite.

28. 28. The vaccine of claim 27, wherein the virus is selected from a DNA virus or an RNA virus, the virus is selected from the Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae families, and the virus is selected from one or more of the novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

29. 29. The vaccine of claim 28, wherein the virus is selected from a novel coronavirus and / or an influenza virus.

30. 28. The vaccine of claim 27, wherein the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.

31. 20. A method for preparing a vaccine, comprising thoroughly mixing an adjuvant composition according to claim 13 and / or a vaccine adjuvant according to claim 19 with a vaccine antigen to obtain said vaccine.

32. 32. The preparation method of claim 31, wherein the vaccine antigens include inactivated or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.

33. 33. The method of claim 32, wherein the pathogen comprises a virus, a bacterium and / or a parasite.

34. 34. The method of claim 33, wherein the pathogen comprises a virus and / or a parasite.

35. The preparation method according to claim 34, wherein the virus is selected from DNA viruses or RNA viruses, the virus is selected from Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae, and the virus is selected from one or more of the novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

36. The preparation method according to claim 35, wherein the virus is selected from a novel coronavirus and / or an influenza virus.

37. 35. The method of claim 34, wherein the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.

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