WO2023131356A2 - 四氧化三锰颗粒在制备疫苗佐剂中的应用 - Google Patents

四氧化三锰颗粒在制备疫苗佐剂中的应用 Download PDF

Info

Publication number
WO2023131356A2
WO2023131356A2 PCT/CN2023/079905 CN2023079905W WO2023131356A2 WO 2023131356 A2 WO2023131356 A2 WO 2023131356A2 CN 2023079905 W CN2023079905 W CN 2023079905W WO 2023131356 A2 WO2023131356 A2 WO 2023131356A2
Authority
WO
WIPO (PCT)
Prior art keywords
adjuvant
vaccine
virus
trimanganese tetraoxide
particle
Prior art date
Application number
PCT/CN2023/079905
Other languages
English (en)
French (fr)
Other versions
WO2023131356A3 (zh
Inventor
王亚玲
陈春英
赵宇亮
Original Assignee
广东粤港澳大湾区国家纳米科技创新研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东粤港澳大湾区国家纳米科技创新研究院 filed Critical 广东粤港澳大湾区国家纳米科技创新研究院
Priority to AU2023205611A priority Critical patent/AU2023205611A1/en
Priority to IL314123A priority patent/IL314123A/en
Publication of WO2023131356A2 publication Critical patent/WO2023131356A2/zh
Publication of WO2023131356A3 publication Critical patent/WO2023131356A3/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • the invention relates to the fields of biomedical technology and vaccine technology, in particular to the application of trimanganese tetraoxide particles in the preparation of vaccine adjuvants.
  • Vaccines refer to biological products made from various pathogenic microorganisms for vaccination. Since the development of vaccines, many diseases such as smallpox have been eliminated. Various types of vaccines have been developed to combat infectious diseases such as the new coronavirus, such as nucleic acid vaccines, inactivated virus vaccines, recombinant protein vaccines, viral vector vaccines, subunit vaccines, etc. Among them, subunit vaccines have become the most researched vaccines due to their good safety, wide use, and high customization. However, the immunogenicity of subunit vaccines is weak, so adjuvants are needed to assist repeated administration. As a non-specific immune enhancer, adjuvants play an important role in inducing an effective immune response after vaccination.
  • Aluminum adjuvants can induce effective humoral immune response, but it is difficult to induce cellular immune response, and more and more evidence shows that antibody and T cell-mediated immunity are required to effectively defend against the new coronavirus.
  • aluminum adjuvants can only activate humoral immunity and lack mucosal immunity.
  • Manganese is a nutritive inorganic trace element required for a variety of physiological processes, including development, reproduction, neuronal function, and more. In recent years, the role of manganese as an immune stimulant has gradually been discovered. Manganese adjuvant can induce the production of type I interferon and cytokines without any infection; in addition, manganese can activate cGAS-STING pathway and induce humoral and cellular immune responses. At present, there have been reports on the application of divalent manganese and tetravalent manganese in vaccine adjuvants.
  • the Chinese patent discloses the use of divalent manganese in the preparation of drugs for improving innate immunity or/or adaptive immunity; and a manganese composition containing divalent manganese for immune enhancement; and a divalent manganese composition Manganese oxide nano adjuvant and its preparation method and application.
  • the immune enhancement effect of pure divalent manganese adjuvant or tetravalent manganese adjuvant needs to be improved.
  • CPG ODN CpG oligonucleotide, CpG oligodeoxynucleotide
  • ODN oligodeoxynucleotide
  • CpG cytosine guanine dinucleotide
  • TLR9 cytosine guanine dinucleotide
  • the Chinese patent discloses the immune effect of the CpG-enhanced divalent manganese adjuvant. When the adjuvant is applied to the new crown subunit vaccine, the amount of Mn element and CpG required is relatively high (Emerg Microbes Infect 2021, 10(1), 1555-1573.).
  • the purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and provide the application of trimanganese tetraoxide particles in the preparation of vaccine adjuvants.
  • the trimanganese tetraoxide particle adjuvants provided by the present invention can effectively combine single-stranded nucleotide adjuvant and can effectively carry the immune antigen to obtain the adjuvant composition vaccine (Fig. Antigens are delivered to lymph node tissue, highly efficient activation of immune cells, and balanced humoral and cellular immunity.
  • the first object of the present invention is to provide an application of trimanganese tetraoxide particles in the preparation of vaccine adjuvants.
  • the second object of the present invention is to provide an adjuvant composition.
  • the third object of the present invention is to provide a vaccine adjuvant.
  • the fourth object of the present invention is to provide a vaccine.
  • the fifth object of the present invention is to provide a preparation method of the vaccine.
  • the adjuvant is a granular adjuvant
  • the adjuvant is a granular adjuvant
  • the granular adjuvant is an externally coated manganese tetraoxide with or without an excipient Trimanganese particles
  • the particle size of the particle adjuvant is 5-3000nm.
  • the trimanganese tetroxide particles are coated with excipients, and the excipients are one or more of proteins, polypeptides, polymers, nucleic acids, and polysaccharides, and the excipients can be combined with the The single-stranded nucleotide adjuvant reacts by non-covalent adsorption or chemoselective covalent modification such that the excipient is coupled to the single-stranded nucleotide adjuvant.
  • the single-stranded nucleotide adjuvant is an oligonucleotide containing CpG ODN.
  • the oligonucleotide is a DNA fragment, ATP, ADP, or AMP.
  • the chemoselective covalent modification is based on the pair of chemoselective covalent modification groups carried by the excipient and the single-stranded nucleotide adjuvant, and the chemoselective covalent modification group Groups include: maleimide with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, succinimide with 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide.
  • the polypeptide is an extracted polypeptide composed of different amino acid sequences, or a chemically synthesized polypeptide.
  • the polymer may be polyethylene glycol, polydopamine, or a polymer with amino or carboxyl groups on the surface of polyethylenediamine.
  • the polysaccharide comprises starch, glycogen, cellulose, chitin, inulin, agar, or hyaluronic acid.
  • the molar ratio of manganese element to excipient in trimanganese tetraoxide particles is (20-4000):1.
  • the molar ratio of manganese element to excipient in trimanganese tetraoxide particles is (20-400):1.
  • the molar ratio of manganese element to excipient in trimanganese tetraoxide particles is (20-300):1.
  • the preparation method of the trimanganese tetraoxide particles is as follows: a divalent manganese salt solution and an excipient molecular solution are fully mixed to obtain to a mixed solution, mixed with an alkaline solution, fully reacted, and purified by dialysis to obtain trimanganese tetraoxide particles.
  • the pH value of the solution is 6-14.
  • the pH value of the solution is 6.5-7.4.
  • particle adjuvants of different sizes can be obtained by changing the molar ratio between Mn 2+ and excipients and OH- .
  • one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia water, triethylamine, pyridine, N-methylmorpholine, and tetramethylethylenediamine are used to adjust the pH of the mixed solution.
  • the concentration of the divalent manganese salt in the mixed solution is 0.01-0.5 mmol/mL, and the concentration of the excipient is 0.005-0.000125 mmol/mL.
  • the conditions for thorough mixing are: the temperature is 30-37° C., and the reaction is stirred for 0.2-5 hours.
  • the conditions for sufficient reaction are: react at a temperature of 30-37° C. for 0.2-5 hours, and then raise the temperature to 60-95° C. and stir for 0.2-5 hours to promote the growth and stability of the crystal form.
  • an adjuvant composition contains a granule adjuvant and a single-chain nucleotide adjuvant, and the granule adjuvant is the trimanganese tetraoxide granules with or without excipients.
  • the single-stranded nucleotide adjuvant is an oligonucleotide containing CpG ODN.
  • the oligonucleotide is a DNA fragment, ATP, ADP, or AMP.
  • the DNA fragments are modified DNA fragments or unmodified DNA fragments.
  • the modified DNA fragments are functionalized with amino, carboxyl, or sulfhydryl functional groups, and chemoselective covalent modification groups on DNA fragments, including maleimidization and succinimidization , 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride functional modification, in order to realize the specific binding of modified DNA on the surface of trimanganese tetraoxide particles.
  • the CpG ODN is divided into A type, B type, and C type according to its immune-induced strong Th1 response and cellular immunostimulatory activity:
  • A-type CpG ODN induces plasmacytoid dendritic cells (pDC) to produce a large amount of IFN- ⁇ , a weak stimulator of NF- ⁇ B;
  • type B CpG ODNs strongly activate B cells but weakly stimulate IFN- ⁇ secretion;
  • type C CpG ODNs combine features of types A and B.
  • Type C CpG ODN induces robust IFN- ⁇ production from pDC and B cell stimulation.
  • the single-stranded nucleotide is tcgtcgttttcggcgcgcgccg-SH.
  • a vaccine adjuvant which is prepared by coupling the particle adjuvant and the single-stranded nucleotide adjuvant in the adjuvant composition in a buffer solution with a pH of 6-9 through a chemoselective covalent modification reaction. Combined for 0.5 to 24 hours, it can be obtained after purification.
  • the granular adjuvant is trimanganese tetroxide particles coated with excipients, and the granular adjuvant is coupled with a single-stranded nucleotide adjuvant through covalent modification; the granular adjuvant is not coated with excipients Trimanganese tetroxide particle adjuvant through adsorption and single-stranded nucleotide adjuvant combination of agents.
  • the chemical selective covalent modification reaction coupling is to activate the functional group of the particle adjuvant and the single-stranded nucleotide adjuvant in the adjuvant composition, and then use the activated functional group
  • the particle adjuvant and the single-stranded nucleotide adjuvant are coupled through a chemoselective covalent modification reaction.
  • the functional group activation of the granular adjuvant includes N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide activating amino or sulfhydryl groups, adjusting pH to change the spatial structure of the excipient;
  • the single-stranded nucleotide functional group activation includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to activate carboxyl or phospholipid groups.
  • the particle adjuvant and the single-stranded nucleotide are coupled by EDC/NHS reaction, electrostatic or coordination adsorption.
  • the molar ratio of manganese element to the nucleotide adjuvant in the adjuvant composition is 1: (0.001-1000).
  • a vaccine containing the adjuvant composition and/or the vaccine adjuvant is provided.
  • the adjuvant composition and/or the vaccine adjuvant carries vaccine antigens.
  • the vaccine antigens include inactivated pathogens or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.
  • the pathogens include viruses, bacteria and/or parasites.
  • the pathogens include viruses and/or parasites.
  • the virus is selected from DNA virus or RNA virus; specifically, the virus is selected from: Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae Viridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae, more preferably said virus is selected from: novel coronavirus, influenza virus, simple One or more of herpes virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV.
  • the virus is selected from: novel coronavirus and/or influenza virus.
  • the bacteria are selected from Gram-positive bacteria and Gram-negative bacteria, specifically, the bacteria are selected from: Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis , Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter Baumea, Mycobacterium tuberculosis, and one or more of Helicobacter pylori.
  • the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.
  • a preparation method of a vaccine wherein the adjuvant composition and/or the vaccine adjuvant is fully mixed with a vaccine antigen to obtain the vaccine.
  • the method of thorough mixing is to repeatedly pump the syringe 50 to 200 times, and after mixing evenly, place it on a rotary shaker Mix for 10-60 minutes.
  • the vaccine antigens include inactivated pathogens or extracted pathogen subunit antigens, recombinant subunit antigens, antigenic epitope peptides, nucleic acid antigens and combinations thereof.
  • said pathogens include viruses, bacteria and/or parasites.
  • the pathogens include viruses and/or parasites.
  • the virus is selected from DNA virus or RNA virus; specifically, the virus is selected from: Coronaviridae, Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae Viridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae, more preferably said virus is selected from: novel coronavirus, influenza virus, simple One or more of herpes virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV.
  • the virus is selected from: novel coronavirus and/or influenza virus.
  • the bacteria are selected from Gram-positive bacteria and Gram-negative bacteria, specifically, the bacteria are selected from: Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis , Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter Baumea, Mycobacterium tuberculosis, and one or more of Helicobacter pylori.
  • the parasite is selected from one or more of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria and Leishmania.
  • the administration method of the adjuvant vaccine combination reagent includes intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, mucosal administration and any combination thereof.
  • the present invention has the following beneficial effects:
  • trimanganese tetroxide particles provided by the present invention can effectively bind single-stranded nucleotide adjuvants and effectively carry immune antigens, and can obtain more excellent immunotherapy with less antigen dosage and lower injection dosage Effect: Efficient activation of immune cells to achieve balanced humoral and cellular immunity.
  • Figure 1 is a schematic diagram of the structure of an adjuvant composition vaccine
  • X1 can be a protein, polypeptide, polymer, nucleic acid, polysaccharide, and its surface has a modifiable amino, carboxyl or sulfhydryl group; it can also be a protein, polypeptide, or polymer containing Y1 , nucleic acid, polysaccharide
  • Y1 is a chemoselective cross-linking group (such as maleimide, succinimide)
  • ODN1 is a negatively charged single-stranded nucleotide (including CpG, CpG derivatives and other single chain polynucleotide)
  • ODN2 is a single-stranded nucleotide containing Y1, which can be combined with a manganese adjuvant containing X1
  • ODN3 is a single-stranded nucleotide functionalized with amino, carboxyl, and sulfhydryl groups
  • Fig. 2 is a TEM image of trimanganese tetraoxide granular adjuvant obtained in Example 1 and Example 2 of the present invention.
  • Fig. 3 is an XRD image of the trimanganese tetraoxide particle adjuvant obtained in Example 1 of the present invention.
  • Fig. 4 is the adjuvant composition vaccine constructed in Example 4 of the present invention and different experimental group vaccine BMDCs activation and antigen presentation ability evaluation in Example 5, Fig. 4A is BMDC activation experiment result, B is MHC-I expression result, C For MHC-II expression results.
  • Figure 5 shows the results of antibody levels in serum of mice on 19d, 35d and 56d after immunization with the adjuvant composition vaccine constructed in Example 4 of the present invention and different formulation vaccines in Example 5.
  • Fig. 6 shows the results of antibody levels related to cellular immunity and humoral immunity in the serum of 56d mice immunized with the adjuvant composition vaccine constructed in Example 4 of the present invention and different formulation vaccines in Example 5.
  • Fig. 7 shows the results of pseudovirus neutralizing antibodies in mouse serum after 56 days of immunizing mice with the adjuvant composition vaccine constructed in Example 4 of the present invention and different formulation vaccines in Example 5.
  • Figure 8 shows the results of specific antibody titers 2 weeks after secondary immunization of mice with influenza H1N1 HA antigen prepared by MnCpG, Mn2CpG or commercial aluminum adjuvant Alum prepared in Step A and Step B of Example 4 of the present invention.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
  • the new crown RBD antigen was purchased from Yiqiao Shenzhou Company, product number 40592-V08H4;
  • the experimental group CpG adjuvant used in the exemplary embodiment of the present invention is: C-type CpG-ODN 2395, [5'-tcgtcgttttcggcgcgcgccg-3'], InvivoGen Company, product number: tlrl-2395-1.
  • the thiol-modified CpG ODN (sequence: tcgtcgttttcggcgcgcgccg-SH) in the example was custom-synthesized by Sangon Biotech.
  • Carboxylic acid polyethylene glycol maleimide COOH-PEG-MAL (manufacturer product number PEG-MSCM), methoxy polyethylene glycol propionic acid, mPEG-COOH (manufacturer product number mPEG-PA-2K), purchased from Jinpan Bio.
  • the premix solution was added to the NaOH solution at a rate of 6.9 ml/min through a peristaltic pump for mixing, and the pH value of the solution was adjusted to 6.9.
  • Example 2 The preparation method of trimanganese tetraoxide microparticle adjuvant
  • the premix was added to the KOH solution at a rate of 3.1 ml/min by a peristaltic pump for mixing, and the pH of the solution was adjusted to 7.4.
  • the premix was added to the Ca(OH) 2 solution at a speed of 5.8 ml/min through a peristaltic pump for mixing, and the pH value of the solution was adjusted to 6.5.
  • the premix solution was added into the ammonia solution at a rate of 7.2 ml/min by a peristaltic pump for mixing, and the pH value of the solution was adjusted to 7.1.
  • the premix solution was added into the triethylamine solution at a rate of 1.8 ml/min through a peristaltic pump for mixing, and the pH value of the solution was adjusted to 7.0.
  • Example 7 The preparation method of trimanganese tetraoxide nanoparticle adjuvant
  • the premix was added to the triethylamine solution at a rate of 4.8 ml/min by a peristaltic pump for mixing, and the pH of the solution was adjusted to 7.1.
  • Example 8 The preparation method of trimanganese tetraoxide nanoparticle adjuvant
  • the premixed solution was added into the triethylamine solution at a speed of 6.9 ml/min through a peristaltic pump for mixing, and the pH value of the solution was adjusted to 6.7.
  • Example 9 The preparation method of trimanganese tetraoxide nanoparticle adjuvant
  • the premix solution was added into the triethylamine solution at a rate of 3.8 ml/min through a peristaltic pump for mixing, and the pH value of the solution was adjusted to 7.2.
  • the concentration of the trimanganese tetroxide particle adjuvant prepared in Example 1 and Example 2 was diluted to 10 ⁇ g/ml, dropped on a common carbon support membrane, and examined under an electron microscope (FEI Company, model Tecnai G2 20S-TWIN) to observe the structure of trimanganese tetraoxide particle adjuvant.
  • Figure 2 is a TEM image of the trimanganese tetraoxide particles prepared in Example 1 (left figure) and Example 2 (right figure).
  • the trimanganese tetraoxide granular adjuvant prepared in Example 1 and Example 2 are all granular.
  • the average size of the obtained manganese particle adjuvant is 13.1 nanometers
  • the obtained product is a micron-sized particle formed by the aggregation of small particles, and the average particle size is 1582.1 nanometers. nm, distributed in the range of 350-3000nm.
  • the concentration of the trimanganese tetraoxide particle adjuvant prepared in Examples 1-9 of the present invention was diluted to 10 ⁇ g/ml
  • the hydrated particle size of the trimanganese tetraoxide particle adjuvant (purchased from Malvern Company, Zetasizer Nano ZS model) was tested with a nanometer particle size analyzer, and the results are shown in Table 1.
  • Example 2 without excipients are compared with the examples 1 with excipients, and the trimanganese tetraoxide particles prepared in Examples 3-9, because there are In the presence of excipients, the resulting particle size is smaller, the distribution is more uniform, the dispersibility is better, and it is more stable in aqueous solution.
  • A) Mix 5ml of 0.125mmol/ml aqueous solution of manganese tetraoxide particles prepared in Example 1 with an equal volume of 0.0004mmol/ml mercapto-modified CpG (purchased from Sangon Biotechnology, tcgtcgttttcggcgcgcgccg-SH), and maintain the pH 8.5 condition , after reacting for 2 hours under stirring at room temperature at 300rpm, after ultrafiltration and purification, the preparation of the adjuvant composition of trimanganese tetraoxide nanoparticle adjuvant and CpG is completed, and the adjuvant combination of trimanganese tetraoxide nanoparticle adjuvant and CpG is obtained substance (adjuvant composition MnCpG).
  • Step C) Disperse the adjuvant compositions prepared in Steps A and B of this example into normal saline, respectively, add 10 ⁇ g of the recombinant RBD antigen of the new coronavirus (purchased from Yiqiao Shenzhou, product number 40592-V08H4) and repeatedly pump the syringe 100 times After mixing evenly, place on a rotary shaker and mix for 10-60 minutes to obtain an adjuvant composition based on a combination of manganese tetraoxide nanoparticle adjuvant and CpG adjuvant carrying antigen (new crown RBD recombinant subunit) vaccine.
  • the new coronavirus purchased from Yiqiao Shenzhou, product number 40592-V08H4
  • 1Ctr control group: the volume of the injection is 100 microliters, and the injection is normal saline;
  • 2Ag group the injection volume is 100 ⁇ l, and 10 ⁇ g RBD antigen is injected;
  • CpG-Ag group the injection volume is 100 microliters, inject 10 ⁇ g RBD antigen and CpG-ODN 2395 (purchased from Invivogen Company, InvivoGen Company, product number: tlrl-2395-1) adjuvant compound group;
  • 4Mn-Ag group the volume of the injection was 100 microliters, and the manganese element containing 25 ⁇ g manganese tetraoxide nanoparticle adjuvant prepared in Example 1 of the present invention carrying 10 ⁇ g RBD antigen (Ag) was injected;
  • 5Alum-Ag group the injection volume was 100 microliters, and 50 ⁇ g commercial aluminum adjuvant Alum (Invivogen Company, Inc., Invivogen, Inc., adjuvant 2%, CAS: 21645-51-2);
  • 6MnCpG-Ag group the volume of the injection was 100 microliters, and the adjuvant composition vaccine constructed in step A of Example 4 was injected, that is, the adjuvant of trimanganese tetraoxide nanoparticle adjuvant and CpG loaded with 10 ⁇ g of RBD antigen (Ag). adjuvant composition (adjuvant composition MnCpG).
  • BMDC cells were seeded in 6-well plates at 3 ⁇ 105 cells per well and allowed to grow overnight. Then the materials of the above six different experimental groups were added, and after further incubation for 24 hours, the cells were collected and stained with anti-CD11c, anti-CD80, anti-CD86, anti-MHC-1, and anti-MHC-II flow staining solutions. The expression levels of co-stimulatory factors CD80, CD86 and antigen recognition signals MHC-1 and MHC-II on the surface of BMDCs were measured by flow cytometry.
  • Figure 4 is the evaluation of BMDCs activation and antigen presentation ability of the vaccine prepared with the adjuvant composition MnCpG constructed in Example 11 of the present invention and other vaccines with different formulations in Example 6.
  • Figure 4A is the result of BMDC activation experiment
  • B is the result of MHC-I expression
  • C is the result of MHC-II expression.
  • the adjuvant composition vaccine constructed in Example 11 has a better BMDCs 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 BMDC activation of about 30.4, which is 2.28 times that of the simple CpG group; and in terms of antigen presentation ability, the MnCpG-Ag group also shows better than the simple CpG Antigen presentation effect.
  • 1Ctr group control group: the volume of the injection is 100 microliters, and the injection is normal saline;
  • 2Ag group the injection volume is 100 microliters, and 10 ⁇ g RBD antigen is injected;
  • 3 CpG-Ag group the injection volume is 100 microliters, inject 10 ⁇ g RBD antigen and CpG-ODN 2395 (purchased from Invivogen Company, InvivoGen Company, product number: tlrl-2395-1) adjuvant complex;
  • 4Mn-Ag group the volume of the injection was 100 microliters, and the nano-vaccine was constructed by injecting 25 ⁇ g of manganese element containing 25 ⁇ g of manganese tetraoxide particle adjuvant prepared in Example 1 of the present invention carrying 10 ⁇ g of RBD antigen (Ag);
  • 5Alum-Ag group the injection volume was 100 microliters, and 50 ⁇ g commercial aluminum adjuvant Alum (Invivogen Company, Inc., Invivogen, Inc., adjuvant 2%, CAS: 21645-51-2);
  • 6MnCpG-Ag group the injection volume is 100 microliters, and the adjuvant composition vaccine constructed in step A of Example 11 is injected, that is, the adjuvant combination of trimanganese tetraoxide nanoparticles carrying 10 ⁇ g of RBD antigen (Ag) and CpG substance (adjuvant composition MnCpG).
  • 7Mn2CpG-Ag group the volume of the injection is 100 microliters, and the vaccine constructed in step B of Example 11 is injected, which is the adjuvant composition (adjuvant) of trimanganese tetraoxide microparticles and CpG carrying 10 ⁇ g of RBD antigen (Ag).
  • composition Mn2CpG the adjuvant composition of trimanganese tetraoxide microparticles and CpG carrying 10 ⁇ g of RBD antigen (Ag).
  • mice aged 6-8 weeks were selected to be vaccinated twice, and the 6 vaccines in the above groups were injected intramuscularly to immunize mice, 100 microliters/mouse, each group The number of mice is 5, and there are seven groups in total. The first inoculation was taken as day 0, the second inoculation was taken as day 21, and serum samples were collected on days 19, 35 and 56.
  • IgG titer levels in serum of mice induced by different experimental groups of vaccines in step A were evaluated by traditional enzyme-linked immunosorbent assay (ELISA).
  • the mouse serum was serially diluted; the diluted serum was added to the RBD antigen pre-coated (2 ⁇ g/ml) 96-well microtiter plate, and stood at 37°C for 2h; after washing, the diluted HRP-conjugated Goat anti-mouse IgG antibody (dilution 1:2000), 100 ⁇ l per well. Stand at 37°C for 1.5h; add TMB chromogenic solution after washing, incubate together, add stop solution to stop the reaction; use a microplate reader to read the absorbance at OD450.
  • the mouse serum was collected 56 days after the immunization in Step A of Example 6 of the present invention, and the level of vaccine-induced cellular immunity and humoral immunity-biased antibodies was evaluated.
  • Figure 5 shows the serum RBD-specific IgG antibody titers of mice 19, 35 and 56 days after the adjuvant composition vaccine (MnCpG-Ag group and Mn2CpG-Ag group) constructed in Example 4 was immunized with other groups in this example.
  • the adjuvant composition vaccines constructed in Example 11 can effectively enhance the antibody response induced by the new crown vaccine in mice.
  • the adjuvant composition vaccines (MnCpG-Ag group and Mn2CpG-Ag group) constructed in Example 11 can effectively enhance the level of IgG immune response.
  • the antibody titer level of the adjuvant composition vaccine group (MnCpG-Ag group and Mn2CpG-Ag group) constructed in Example 8 was the same as that of simple trimanganese tetraoxide particles.
  • Adjuvant Mn-Ag group vaccine is 4 times, 10 times that of pure Alum adjuvant vaccine antibody, and 50 times that of simple CpG adjuvant group vaccine antibody; it shows that the formation of adjuvant combination with CpG can improve the level of trimanganese tetraoxide particle adjuvant immune potentiating ability.
  • the antibody produced by the MnCpG-Ag group in Example 12 is 2.2 times that of the Mn2CpG group, indicating that the nanoparticle adjuvant bound to CpG by covalent modification can produce a micron particle vaccine bound to CpG by covalent modification Higher levels of antibodies were produced.
  • the antibody level of the Mn2CpG-Ag group is 2 times that of the Mn-Ag group, showing that even if the adsorption mode is used to prepare the antibody produced by the adjuvant composition, it is also higher than the group of the simple trimanganese tetraoxide adjuvant; it can be seen that no matter the adsorption (implementation Example 2) or the combination of covalent modification (Example 1) scheme combination CpG and trimanganese tetraoxide particle adjuvant can significantly improve the respective vaccine synergistic ability, compared with pure antigen Ag group and CpG-Ag group to promote more High antibody production levels.
  • FIG. 6 is the evaluation of the effect of the adjuvant composition vaccine constructed in Example 11 of the present invention and the different formula vaccines in Example 12 in inducing cellular and humoral immune balance.
  • the serum samples of each group of mice were subjected to a neutralization test for pseudovirus infection.
  • the specific process was as follows: The supernatant was pre-incubated with serially diluted mouse serum for 1 hour at 37°C and then added to ACE2-expressing 293T cells (5 ⁇ 10 4 cells). Fresh medium was added after 24 hours and cells were lysed using commercially available cell lysis buffer. Relative luciferase activity was determined in a luminometer (Bio-Tech) after addition of luciferase substrate. The neutralization efficiency of the pseudovirus was calculated and expressed as 50% neutralizing antibody titer.
  • Fig. 7 is the adjuvant composition vaccine constructed in Example 11 and other formula vaccines constructed in Example 12 Comparison of in vitro immune effects of vaccines against pseudoviruses. It can be seen from Figure 7 that the vaccines prepared by the adjuvant compositions MnCpG and Mn2CpG obtained in Example 11 can induce a significantly enhanced neutralizing antibody response against the novel coronavirus. However, the neutralization ability of MnCpG-Ag and Mn2CpG-Ag group sera to the new coronavirus is equivalent.
  • the prepared adjuvant composition influenza subunit vaccines MnCpG-HA and Mn2CpG-HA were immunized by intramuscular injection, and the titers of antigen-specific IgG and IgM antibodies produced were determined.
  • mice Balb/C mice, 6-8 weeks old, 5 mice/group, female.
  • H1N1 HA antigen 5 ⁇ g/mouse 5 ⁇ g/mouse
  • commercial aluminum adjuvant Alum 100 ⁇ g/100 ⁇ l/mouse
  • MnCpG-HA and Mn2CpG-HA 100 ⁇ g/100 ⁇ l/mouse ;
  • HA group H1N1 HA antigen 5 ⁇ g/mouse
  • Alum-HA group commercial aluminum adjuvant Alum 100 ⁇ g/100 ⁇ l+H1N1 HA 5 ⁇ g/mouse
  • MnCpG-HA group 100 ⁇ l injection, containing MnCpG 100 ⁇ g and H1N1 HA antigen 5 ⁇ g/mouse
  • Mn2CpG-HA group 100 ⁇ l injection, containing Mn2CpG 100 ⁇ g and H1N1 HA antigen 5 ⁇ g/mouse.
  • mice were immunized with vaccines in each group, the mice were boosted 3 weeks after the first immunization according to the immunization group, and 2 weeks after the second immunization, blood was collected from the orbit to measure the antibody titer in the serum.
  • Antibody titers in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IgG and IgM induced by the vaccine in mouse serum were evaluated.
  • ELISA enzyme-linked immunosorbent assay
  • Fig. 8 is the specificity produced after the adjuvant composition MnCpG, the adjuvant composition Mn2CpG or the commercial aluminum adjuvant Alum prepared in Step A and Step B of Example 11 of the present application carry the influenza H1N1 HA antigen to immunize mice Antibody titer analysis results.
  • the vaccines prepared with the adjuvant composition MnCpG and the adjuvant composition Mn2CpG obtained in Steps A and B of Example 11 of the present invention have higher IgM levels than vaccines prepared with commercial aluminum adjuvants, producing The antibody titers of the aluminum adjuvant were 4.3 times and 3.1 times respectively.
  • the aqueous solution of the trimanganese tetraoxide particles prepared by the embodiment 6 of 5ml 0.2mmol/ml is mixed with an equal volume 0.0005mmol/ml thiol-modified CpG (purchased from Sangon Biotechnology, tcgtcgttttcggcgcgcgccg-SH) was mixed, maintained at pH 8.4, reacted at room temperature for 2 hours under stirring at 500rpm, and purified by ultrafiltration to complete the trimanganese tetraoxide nanoparticle adjuvant
  • the preparation of the adjuvant composition of agent and CpG obtains the adjuvant composition (adjuvant composition Mn6CpG) of trimanganese tetraoxide nanoparticle adjuvant and CpG.
  • the novel dense granule protein GRA is one of the main components of excretory-secretory antigens (ESAs) secreted by the parasite when it actively invades host cells, and is related to the intracellular maintenance of the parasite. Relevant studies have shown that it can be used as a vaccine drug.
  • ESAs excretory-secretory antigens
  • mice were randomly divided into groups of 10:
  • 1Ctr group control group: the volume of the injection is 100 microliters, and the injection is normal saline;
  • 2Ag group the injection volume is 100 ⁇ l, and 10 ⁇ g of GRA antigen is injected;
  • 3ATP-Ag group the injection volume is 100 microliters, and 10 ⁇ g of GRA antigen is injected with ATP adjuvant;
  • 4ADP-Ag group the injection volume is 100 microliters, inject 10 ⁇ g of GRA antigen and ADP adjuvant;
  • 5Mn3-Ag group the volume of the injection was 100 microliters, and the nano-vaccine constructed by the manganese element containing 25 ⁇ g manganese tetraoxide particle adjuvant prepared in Example 3 of the present invention carrying 10 ⁇ g GRA antigen was injected;
  • 6Mn4-Ag group the volume of injection was 100 microliters, and 50 ⁇ g commercial aluminum adjuvant Alum (Invivogen Company, Inc., Invivogen, Inc.) was injected with 10 ⁇ g RBD antigen adjuvant 2%, CAS: 21645-51-2);
  • 7Mn3ATP-Ag group the volume of injection was 100 microliters, and the adjuvant composition vaccine constructed in Step A of Example 16 was injected, that is, Mn3ATP carrying 10 ⁇ g of RBD antigen (Ag).
  • 8Mn4ADP-Ag group the volume of injection was 100 microliters, and the vaccine constructed in Step B of Example 16 was injected, that is, Mn4ADP carrying 10 ⁇ g of RBD antigen (Ag).
  • the adjuvant composition prepared in Example 16 was mixed with 10 micrograms of the GRA vaccine drug respectively, and compared with the group without the adjuvant composition, the experimental mice were immunized. Five weeks after the injection, the mice in each experimental group were infected with Toxoplasma gondii RH strain. Observe and record the survival rate of the mice in each test group every day, and the experimental results are shown in the table below:
  • trimanganese tetraoxide nano-adjuvants constructed in Examples 3 and 4 and the combined nano-adjuvants Mn3ATP and Mn4ADP constructed in Example 16 can significantly enhance the immune effect of the original GRA vaccine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明公开了四氧化三锰颗粒在制备疫苗佐剂中的应用，所述佐剂为颗粒佐剂，所述颗粒佐剂为外部包裹有或没有赋形剂的四氧化三锰颗粒，所述颗粒佐剂粒径为5～3000nm。本发明所提供的四氧化三锰颗粒佐剂能够有效结合单链核苷酸佐剂并能有效载带免疫抗原，能够在较少的抗原剂量和较低的注射使用量时，获得更加优异的免疫治疗效果；高效的激活免疫细胞，实现平衡的体液和细胞免疫。

Description

四氧化三锰颗粒在制备疫苗佐剂中的应用 技术领域
本发明涉及生物医药技术与疫苗技术领域，具体地，涉及四氧化三锰颗粒在制备疫苗佐剂中的应用。
背景技术
疫苗是指用各类病原微生物制作的用于预防接种的生物制品。自疫苗发展以来，已实现对天花等多种疾病的消除。多种类型的疫苗已被研发用于抗击新冠病毒等传染性疾病，如核酸疫苗、灭活病毒疫苗、重组蛋白疫苗、病毒载体疫苗、亚单位疫苗等。其中，亚单位疫苗因其安全性好、使用广泛、高度可定制，成为目前研究最多的疫苗。但亚单位疫苗的免疫原性较弱，因此需要佐剂辅助重复给药。佐剂作为非特异性免疫增强剂，对疫苗接种后诱导有效的免疫应答，发挥重要的作用。
传统的铝佐剂可诱导有效的体液免疫反应，但难以诱导细胞免疫反应，越来越多的证据表明需要抗体和T细胞介导的免疫才能有效抵御新型冠状病毒。然而，铝佐剂仅能激活体液免疫，缺乏粘膜免疫能力。
锰是多种生理过程所需的营养无机微量元素，包括发育、繁殖、神经元功能等。近年来，锰作为免疫刺激剂的作用也逐渐被发现。锰佐剂可在无任何感染的情况下诱导Ⅰ型干扰素和细胞因子的产生；另外锰还可以激活cGAS-STING通路，诱导体液和细胞免疫反应。目前，已有关于将二价锰和四价锰应用于疫苗佐剂的报道。中国专利公开了一种二价锰在制备用于改善固有免疫或/或适应性免疫的药物中的用途；以及一种用于免疫增强的、包含二价锰的锰组合物；以及一种二氧化锰纳米佐剂及其制备方法、应用。但单纯的二价锰佐剂或四价锰佐剂的免疫增强效果还有待提升。
CPG ODN(CpG oligonucleotide,CpG寡脱氧核苷酸)是人工合成的含有非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)的寡脱氧核苷酸(ODN)，可模拟细菌DNA刺激多种哺乳动物包括人的免疫细胞。其通过内吞作用进入，被TLR9识别并结合，激活NF-κB通路，产生多种细胞因子，增强抗原的加工递呈，诱导Th1免疫反应。中国专利公开了CpG增强的二价锰佐剂的免疫效果，该佐剂在应用于新冠亚单位疫苗时，所需Mn元素以及CpG的用量都较高(Emerg Microbes Infect 2021,10(1),1555-1573.)。
发明内容
本发明的目的是为了克服现有技术的上述不足，提供四氧化三锰颗粒在制备疫苗佐剂中的应用，本发明所提供的四氧化三锰颗粒佐剂能够有效结合单链核苷酸佐剂并能有效载带免疫抗原，得到佐剂组合物疫苗(图1)，能够在较少的抗原载带量和较低的注射使用量时，获得更加优异的免疫治疗效果；能够有效将免疫抗原递送至淋巴结组织，极高效的激活免疫细胞，实现平衡的体液和细胞免疫。相对于现有技术，本发明四氧化三锰颗粒佐剂在使用量为其五分之一，Mn元素以及CpG浓度为其四分之一时，即可获得了高效的靶向递送效果以及优异的免疫激活效果。
本发明的第一个目的是提供一种四氧化三锰颗粒在制备疫苗佐剂中的应用。
本发明的第二个目的是提供一种佐剂组合物。
本发明的第三个目的是提供一种疫苗佐剂。
本发明的第四个目的是提供一种疫苗。
本发明的第五个目的是提供一种疫苗的制备方法。
为了实现上述目的，本发明是通过以下方案予以实现的：
四氧化三锰颗粒在制备疫苗的佐剂中的应用，所述佐剂为颗粒佐剂，所述佐剂为颗粒佐剂，所述颗粒佐剂为外部包裹有或没有赋形剂的四氧化三锰颗粒，所述颗粒佐剂粒径为5～3000nm。
优选地，所述四氧化三锰颗粒外部包裹有赋形剂，所述赋形剂为蛋白、多肽、聚合物、核酸、多糖中的一种或几种，所述赋形剂能够与所述单链核苷酸佐剂通过非共价吸附或化学选择性共价修饰进行反应，使得所述赋形剂与所述单链核苷酸佐剂偶联。
更优选地，所述单链核苷酸佐剂为含有CpG ODN的寡核苷酸。
进一步优选地，所述寡核苷酸为DNA片段、ATP、ADP、或AMP。
进一步优选地，所述化学选择性共价修饰是基于所述赋形剂与所述单链核苷酸佐剂所携带化学选择性共价修饰基团对，所述化学选择性共价修饰基团包括：马来酰亚胺与1-(3-二甲氨基丙基)-3-乙基碳二亚胺、琥珀酰亚胺与1-(3-二甲氨基丙基)-3-乙基碳二亚胺。
更优选地，所述多肽为是由不同氨基酸序列构成的提取的多肽、或化学合成的多肽。
更优选地，所述聚合物可以是聚乙二醇、聚多巴胺、或聚乙二胺表面具有氨基或羧基的聚合物。
更优选地，所述多糖包括淀粉、糖原、纤维素、几丁质、菊糖、琼脂、或透明质酸。
更优选地，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～4000)：1。
进一步优选地，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～400)：1。
更一步优选地，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～300)：1。
优选地，所述四氧化三锰颗粒的制备方法为：二价锰盐水溶液与赋形剂分子溶液充分混合得 到混合溶液，与碱性的溶液混合，充分反应，透析纯化，得到四氧化三锰颗粒。
更优选地，碱性的溶液混合后，溶液pH值为6～14。
进一步优选地，碱性的溶液混合后，溶液pH值为6.5～7.4。
本发明可以通过改变Mn2+与赋形剂、OH-之间的摩尔比可得到不同尺寸的颗粒佐剂。
更优选地，使用氢氧化钠、氢氧化钾、氢氧化钙、氨水、三乙胺、吡啶、N-甲基吗啉、四甲基乙二胺中的一种或多种调节混合溶液pH。
更优选地，混合溶液中二价锰盐的浓度为0.01～0.5mmol/mL，赋形剂的浓度为0.005～0.000125mmol/mL。
更优选地，充分混合的条件为：温度为30～37℃，搅拌反应0.2～5小时。
更优选地，充分反应的条件为：先于温度30～37℃下反应0.2～5小时，后升高温度至60～95℃搅拌反应0.2～5小时，促进晶型生长与稳定。
一种佐剂组合物，所述佐剂复合物含有颗粒佐剂和单链核苷酸佐剂，所述颗粒佐剂为所述的外部包裹有或没有赋形剂的四氧化三锰颗粒。
优选地，所述单链核苷酸佐剂为含有CpG ODN的寡核苷酸。
更优选地，所述寡核苷酸为DNA片段、ATP、ADP、或AMP。
更优选地，所述DNA片为改性DNA片段或未改性DNA片段。
进一步优选地，所述改性DNA片段为对DNA片段进行氨基、羧基、或巯基官能团功能化、化学选择性的共价修饰基团功能化，包括马来酰亚胺化、琥珀酰亚胺化、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐功能化修饰，以实现改性DNA特异性结合于四氧化三锰颗粒的表面。
其中，所述CpG ODN按照其免疫诱导强Th1型应答和细胞免疫刺激活性不同分为A型、B型、和C型：A型CpG ODN诱导浆细胞样树突细胞(pDC)产生大量IFN-α，NF-κB的弱刺激物；B型CpG ODN强烈激活B细胞，但微弱地刺激IFN-α分泌；C型CpG ODN结合了A型和B型的特征。C型CpG ODN从pDC和B细胞刺激中诱导强烈的IFN-α产生。
作为一个具体的实施例，所述单链核苷酸为tcgtcgttttcggcgcgcgccg-SH。
一种疫苗佐剂，其制备方法为将所述的佐剂组合物中的颗粒佐剂和单链核苷酸佐剂在pH为6～9的缓冲液中通过化学选择性共价修饰反应偶联0.5～24小时，经纯化即得。
所述颗粒佐剂为外部包裹有赋形剂的四氧化三锰颗粒，颗粒佐剂通过共价修饰与单链核苷酸佐剂偶联；所述颗粒佐剂为外部没有包裹赋形剂的四氧化三锰颗粒佐剂通过吸附与单链核苷酸佐 剂相结合。
优选地，所述通过化学选择性共价修饰反应偶联为将所述的佐剂组合物中的颗粒佐剂和单链核苷酸佐剂进行功能基团活化，之后利用活化的功能基团将颗粒佐剂和单链核苷酸佐剂通过化学选择性共价修饰反应偶联。
更优选地，将所述颗粒佐剂进行功能基团活化包括N-羟基琥珀酰亚胺(NHS)或N-羟基硫代琥珀酰亚胺活化氨基或巯基、调节pH改变赋形剂空间结构；所述单链核苷酸功能基团活化包括1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)活化羧基或磷脂基团。
优选地，通过EDC/NHS反应、静电或配位作用吸附将所述颗粒佐剂和所述单链核苷酸偶联。
优选地，所述佐剂组合物中锰元素的与所述核苷酸佐剂的摩尔比例为1：(0.001～1000)。
一种疫苗，含有所述的佐剂组合物和/或所述的疫苗佐剂。
优选地，所述的佐剂组合物和/或所述的疫苗佐剂载带疫苗抗原。
更优选地，所述疫苗抗原包括灭活病原体或者提取的病原体亚单位抗原、重组亚单位抗原、抗原表位肽、核酸抗原及其组合。
进一步优选地，所述病原体包括病毒、细菌和/或寄生虫。
更一步优选地，所述病原体包括病毒和/或寄生虫。
更一步优选地，所述病毒选自为DNA病毒或RNA病毒；具体地所述病毒选自：冠状病毒科、疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，更优选地所述病毒选自：新型冠状病毒、流感病毒、单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV中的一种或几种。
更一步优选地，所述病毒选自：新型冠状病毒和/或流感病毒。
更一步优选地，所述细菌选自革兰氏阳性菌和革兰氏阴性菌，具体地，所述细菌选自：肺炎链球菌、流感嗜血杆菌、沙门氏菌、脑膜炎双球菌、表皮葡萄球菌、金黄色葡萄球菌、大肠杆菌、肺炎克雷伯氏菌、产酸克雷伯氏菌、阴沟肠杆菌、弗氏柠檬酸杆菌、绿脓假单胞菌、波美不动杆菌、结核杆菌、和幽门螺杆菌中的一种或几种。
更一步优选地，所述寄生虫选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫中的一种或几种。
一种疫苗的制备方法，所述的佐剂组合物和/或所述的疫苗佐剂与疫苗抗原充分混合，即得。
优选地，所述充分混合的做法为注射器反复抽打50～200下，混合均匀后，置于旋转式摇床 混合10～60分钟。
优选地，所述疫苗抗原包括灭活病原体或者提取的病原体亚单位抗原、重组亚单位抗原、抗原表位肽、核酸抗原及其组合。
更优选地，所述病原体包括病毒、细菌和/或寄生虫。
进一步优选地，所述病原体包括病毒和/或寄生虫。
更一步优选地，所述病毒选自为DNA病毒或RNA病毒；具体地所述病毒选自：冠状病毒科、疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，更优选地所述病毒选自：新型冠状病毒、流感病毒、单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV中的一种或几种。
更一步优选地，所述病毒选自：新型冠状病毒和/或流感病毒。
更一步优选地，所述细菌选自革兰氏阳性菌和革兰氏阴性菌，具体地，所述细菌选自：肺炎链球菌、流感嗜血杆菌、沙门氏菌、脑膜炎双球菌、表皮葡萄球菌、金黄色葡萄球菌、大肠杆菌、肺炎克雷伯氏菌、产酸克雷伯氏菌、阴沟肠杆菌、弗氏柠檬酸杆菌、绿脓假单胞菌、波美不动杆菌、结核杆菌、和幽门螺杆菌中的一种或几种。
优选地，所述寄生虫选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫中的一种或几种。
优选地，所述的佐剂疫苗组合试剂的施用方法，包括肌肉注射、皮下注射、皮内注射、静脉注射、粘膜施用及其任意组合。
与现有技术相比，本发明具有以下有益效果：
本发明所提供的四氧化三锰颗粒能够有效结合单链核苷酸佐剂并能有效载带免疫抗原，能够在较少的抗原剂量和较低的注射使用量时，获得更加优异的免疫治疗效果；高效的激活免疫细胞，实现平衡的体液和细胞免疫。
附图说明
图1为佐剂组合物疫苗的结构示意图；X1可以是蛋白、多肽、聚合物、核酸、多糖，其表面具有可修饰的氨基、羧基或巯基；也可以为含有Y1的蛋白、多肽、聚合物、核酸、多糖；Y1为具有化学选择性的交联基团(如马来酰亚胺、琥珀酰亚胺)；ODN1为带负电的单链核苷酸(包括CpG，CpG衍生物及其他单链聚核苷酸)；ODN2为含有Y1的单链核苷酸，可与含有X1的锰佐剂结合；ODN3为氨基、羧基、巯基功能化的单链核苷酸，可与含Y1的锰佐剂结合。
图2为本发明实施例1和实施例2中获得四氧化三锰颗粒佐剂的TEM图像。
图3为本发明实施例1中获得中四氧化三锰颗粒佐剂的XRD图像。
图4为本发明实施例4所构建的佐剂组合物疫苗与实施例5中不同实验组疫苗BMDCs激活和抗原呈递能力评估，图4A为BMDC激活实验结果，B为MHC-I表达结果，C为MHC-II表达结果。
图5为本发明实施例4所构建的佐剂组合物疫苗与实施例5中不同配方疫苗免疫后小鼠19d，35d和56d血清中的抗体水平结果。
图6为本发明实施例4所构建的佐剂组合物疫苗与实施例5中不同配方疫苗免疫小鼠56d小鼠血清中细胞免疫及体液免疫相关抗体水平结果。
图7为本发明实施例4所构建的佐剂组合物疫苗与实施例5中不同配方疫苗免疫小鼠56d后小鼠血清的假病毒中和抗体结果。
图8为本发明实施例4步骤A和步骤B制备的MnCpG、Mn2CpG或商品铝佐剂Alum载带流感H1N1 HA抗原对小鼠进行二次免疫后2周，产生的特异性抗体滴度结果。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
主要试剂来源：
新冠RBD抗原购自义翘神州公司，货号40592-V08H4；
本发明示例性实施例中采用的实验组CpG佐剂为：C型CpG-ODN 2395，[5’-tcgtcgttttcggcgcgcgccg-3’]，InvivoGen公司，产品货号：tlrl-2395-1。
实施例中巯基化修饰的CpG ODN(序列为：tcgtcgttttcggcgcgcgccg-SH)，由生工生物定制合成。
羧酸聚乙二醇马来酰亚胺，COOH-PEG-MAL(厂商货号PEG-MSCM)，甲氧基聚乙二醇丙酸，mPEG-COOH(厂商货号mPEG-PA-2K)，购自金畔生物。
商品铝佐剂Alum(Invivogen公司，
Figure PCTCN2023079905-ftappb-I100001
adjuvant 2％，CAS:21645-51-2)
实施例1四氧化三锰纳米颗粒佐剂的制备方法
将240ml 0.25mmol/mL可溶性锰盐MnCl2·4H2O的水溶液和1ml 0.02mmol/ml COOH-PEG-MAL与0.18mmol/ml的mPEG-COOH赋形剂混合溶液，充分混合，得到 预混液，其中Mn2+与总赋形剂之间的摩尔比为300：1。
随后，在700rpm搅拌下，通过蠕动泵将预混液，以6.9ml/min速度加入到NaOH溶液中进行混合，调节溶液pH值为6.9。
最终，之后在34℃反应30分钟，升温至80℃反应3h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例2四氧化三锰微米颗粒佐剂的制备方法
将0.5mmol/ml的锰盐MnCl2·4H2O溶于250ml水后，通过蠕动泵以3.9ml/min速度加入到3mmol/ml的NaOH中进行混合反应，结束后继续用0.02M NaOH，调节溶液pH值为6.9。
在500rpm搅拌下、34℃反应30分钟，最后升温至60℃反应3h；反应完成后，静置冷却至室温，离心后加入0.5％氯化钠洗涤，最后高压蒸汽灭菌或经过滤膜除菌。
实施例3四氧化三锰纳米颗粒佐剂的制备方法
将40ml 0.1mmol/mL硫酸锰的水溶液和200ml卵清蛋白赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为200：1。
随后，在1000rpm搅拌下，通过蠕动泵将预混液，以3.1ml/min速度加入到KOH溶液中进行混合，调节溶液pH值为7.4。
最终，之后在30℃反应60分钟，升温至90℃反应2h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例4四氧化三锰纳米颗粒佐剂的制备方法
将200ml 0.2mmol/mL硝酸锰的水溶液和100ml环RGD多肽赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为2：1。
随后，在1400rpm搅拌下，通过蠕动泵将预混液，以5.8ml/min速度加入到Ca(OH)2溶液中进行混合，调节溶液pH值为6.5。
最终，之后在20℃反应90分钟，升温至70℃反应5h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例5四氧化三锰纳米颗粒佐剂的制备方法
将150ml 0.6mmol/mL醋酸锰的水溶液和300ml淀粉(5000Da)赋形剂混合溶液， 充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为100：1。
随后，在900rpm搅拌下，通过蠕动泵将预混液，以7.2ml/min速度加入到氨水溶液中进行混合，调节溶液pH值为7.1。
最终，之后在35℃反应40分钟，升温至80℃反应2h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例6四氧化三锰纳米颗粒佐剂的制备方法
将50ml 0.1mmol/mL氯化锰的水溶液和200ml aptamer核酸适体赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为5：1。
随后，在1400rpm搅拌下，通过蠕动泵将预混液，以1.8ml/min速度加入到三乙胺溶液中进行混合，调节溶液pH值为7.0。
最终，之后在15℃反应90分钟，升温至60℃反应5h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例7四氧化三锰纳米颗粒佐剂的制备方法
将350ml 0.3mmol/mL氯化锰的水溶液和200ml聚乙二胺赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为50：1。
随后，在900rpm搅拌下，通过蠕动泵将预混液，以4.8ml/min速度加入到三乙胺溶液中进行混合，调节溶液pH值为7.1。
最终，之后在35℃反应40分钟，升温至90℃反应2h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例8四氧化三锰纳米颗粒佐剂的制备方法
将450ml 0.2mmol/mL氯化锰的水溶液和300ml几丁质赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为250：1。
随后，在1100rpm搅拌下，通过蠕动泵将预混液，以6.9ml/min速度加入到三乙胺溶液中进行混合，调节溶液pH值为6.7。
最终，之后在25℃反应80分钟，升温至80℃反应2h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例9四氧化三锰纳米颗粒佐剂的制备方法
将250ml 0.5mmol/mL氯化锰的水溶液和200ml糖原赋形剂混合溶液，充分混合，得到预混液，其中Mn2+与总赋形剂之间的摩尔比为50：1。
随后，在1300rpm搅拌下，通过蠕动泵将预混液，以3.8ml/min速度加入到三乙胺溶液中进行混合，调节溶液pH值为7.2。
最终，之后在35℃反应40分钟，升温至90℃反应1h，促进颗粒晶型的生长及稳定；反应完成后，静置冷却至室温后，经离心洗涤，最后高压蒸汽灭菌或通过过滤膜除菌备用。
实施例10四氧化三锰颗粒佐剂的理化性质表征
一、电镜下观察
1、实验方法
(1)25℃条件下，将实施例1和实施例2制备得到的四氧化三锰颗粒佐剂的浓度稀释至10μg/ml，滴在普通碳支持膜上，在电镜(FEI公司，型号Tecnai G2 20S-TWIN)下观察四氧化三锰颗粒佐剂的结构。
2.实验结果
结果如图2所示，图2为实施例1(左图)和实施例2(右图)制备得到的四氧化三锰颗粒的TEM图。
由图2可知，实施例1和实施例2制备得到的四氧化三锰颗粒佐剂均为颗粒状。其中，当有赋形剂存在时，得到的锰颗粒佐剂平均尺寸为13.1纳米，而无赋形剂存在时，得到产物是由小颗粒聚集而成的微米尺寸颗粒，平均粒径尺寸为1582.1nm，分布于350-3000nm的范围内。
二、对所述四氧化三锰颗粒佐剂进行XRD表征
1、实验方法
将有赋形剂存在的实施例1制备得到的四氧化三锰颗粒佐剂取10ml冻干，得到粉末样品，将粉末样品进行XRD表征，之后用Jade进行分析。
2.实验结果
结果如图3所示，结果显示实施例1得到锰颗粒佐剂晶型对应为Mn3O4。
三、对所述四氧化三锰颗粒佐剂的粒径和表面电荷进行表征
1、实验方法
25℃条件下，将本发明实施例1-9制备的四氧化三锰颗粒佐剂的浓度稀释至10μg/ml， 用纳米粒度仪测试四氧化三锰颗粒佐剂的水合粒径(购自Malvern公司，Zetasizer Nano ZS型号)，结果如表1所示。
与TEM结果所显示的类似，无赋形剂存在的实施例2制备得到的颗粒与有赋形剂存在的实施例1，实施例3-9制备得到的四氧化三锰颗粒相比，因为有赋形剂存在时，得到的颗粒尺寸更小，分布更加均匀，分散性更好，在水溶液中更加稳定。
Figure PCTCN2023079905-ftappb-I100002
实施例11四氧化三锰纳米颗粒佐剂与CpG的组合的新冠RBD重组蛋白佐剂组合物疫苗的制作
A)将5ml 0.125mmol/ml的实施例1制备的四氧化三锰颗粒的水溶液与等体积0.0004mmol/ml巯基化修饰的CpG(购自生工生物，tcgtcgttttcggcgcgcgccg-SH)混合，维持pH8.5条件，在室温300rpm搅拌下反应2小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物(佐剂组合物MnCpG)。
B)5ml 0.125mmol/ml的实施例2制备的四氧化三锰颗粒的水溶液与等体积0.0004mmol/ml C型CpG-ODN 2395(5’-tcgtcgttttcggcgcgcgccg-3’)混合，维持pH 8.5条件，在室温300rpm搅拌下反应2小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与CpG-ODN 2395的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物(佐剂组合物Mn2CpG)。
C)将本实施例步骤A和B制得的佐剂组合物分别分散入生理盐水中，并分别加入10μg新冠病毒重组RBD抗原(购自义翘神州，货号40592-V08H4)注射器反复抽打100下，混合均匀后，置于旋转式摇床混合10～60分钟，得到基于四氧化三锰纳米颗粒佐剂与CpG佐剂的组合佐剂载带抗原(新冠RBD重组亚单位)的佐剂组合物疫苗。
实施例12佐剂组合物疫苗的BMDCs激活和抗原呈递能力评估
一、实验方法
1、实验分组
①Ctr(对照组)：注射液体积为100微升，注射液为生理盐水；
②Ag组：注射液体积为100微升，注射10μg RBD抗原；
③CpG-Ag组：注射液体积为100微升，注射10μg RBD抗原与CpG-ODN 2395(购自Invivogen公司，InvivoGen公司，产品货号：tlrl-2395-1)佐剂复合组；
④Mn-Ag组：注射液体积为100微升，注射载带10μg RBD抗原(Ag)的含锰元素25μg本发明实施例1制备的四氧化三锰纳米颗粒佐剂；
⑤Alum-Ag组：注射液体积为100微升，注射载带10μg RBD抗原的50μg商品铝佐剂Alum(Invivogen公司，
Figure PCTCN2023079905-ftappb-I100003
adjuvant 2％，CAS:21645-51-2)；
⑥MnCpG-Ag组：注射液体积为100微升，注射实施例4步骤A所构建的佐剂组合物疫苗，即载带10μg RBD抗原(Ag)的四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物(佐剂组合物MnCpG)。
2、实验方法
将BMDC细胞以每孔3×105个细胞接种在6孔板中，并使其生长过夜。然后分别加入上述6个不同实验组材料，进一步孵育24小时后，收集细胞并用抗CD11c、抗CD80和抗CD86、抗MHC-1、抗MHC-Ⅱ的流式染色液对细胞进行染色。用流式细胞分析仪测定BMDCs表面共刺激因子CD80、CD86和抗原识别信号MHC-1、MHC-Ⅱ的表达水平。
二、实验结果
如图4所示。图4为本发明实施例11所构建的佐剂组合物MnCpG制备的疫苗与实施例6中其他不同配方疫苗BMDCs激活和抗原呈递能力评估。图4A为BMDC激活实验结果，B为MHC-I表达结果，C为MHC-II表达结果。
由图4可知，实施例11所构建的佐剂组合物疫苗与单一佐剂疫苗相比，有更好的BMDCs激活效果，且有更高的抗原呈递能力。本发明实施例1制备的佐剂组合物(MnCpG组)能够刺激约30.4的BMDC激活，是单纯CpG组的2.28倍；而抗原呈递能力方面，MnCpG-Ag组也体现出比单纯CpG更好的抗原呈递效果。
实施例13佐剂组合物疫苗免疫小鼠效果评估
一、实验方法
1、实验分组
①Ctr组(对照组)：注射液体积为100微升，注射液为生理盐水；
②Ag组：注射液体积为100微升，注射10μg RBD抗原；
③CpG-Ag组：注射液体积为100微升，注射10μg RBD抗原与CpG-ODN 2395(购自Invivogen公司，InvivoGen公司，产品货号：tlrl-2395-1)佐剂复合；
④Mn-Ag组：注射液体积为100微升，注射载带10μg RBD抗原(Ag)的含锰元素25μg本发明实施例1制备的四氧化三锰颗粒佐剂构建的纳米疫苗；
⑤Alum-Ag组：注射液体积为100微升，注射载带10μg RBD抗原的50μg商品铝佐剂Alum(Invivogen公司，
Figure PCTCN2023079905-ftappb-I100004
adjuvant 2％，CAS:21645-51-2)；
⑥MnCpG-Ag组：注射液体积为100微升，注射实施例11步骤A所构建的佐剂组合物疫苗，即载带10μg RBD抗原(Ag)的四氧化三锰纳米颗粒与CpG的佐剂组合物(佐剂组合物MnCpG)。
⑦Mn2CpG-Ag组：注射液体积为100微升，注射实施例11步骤B所构建的疫苗，即载带10μg RBD抗原(Ag)的四氧化三锰微米颗粒与CpG的佐剂组合物(佐剂组合物Mn2CpG)。
2、免疫小鼠
在遵循国家动物保健协议的前提下，选取6～8周龄的BALB/c小鼠进行接种2次，将上述分组的6种疫苗，肌肉注射进行小鼠免疫，100微升/鼠，每组小鼠数量为5只，一共有七组。以第一次接种为第0天，第二次接种为第21天，并于第19、35和56天收集血清样品。
B)小鼠血清中IgG滴度的检测
通过传统的酶联免疫吸附测定方法(ELISA)评估步骤A中不同实验组疫苗诱导的小鼠血清中IgG滴度水平。
对小鼠血清进行连续等比稀释；将稀释的血清加到RBD抗原的预包被(2μg/ml)的96孔酶标板中，37℃静置2h；清洗后加入稀释的HRP缀合的山羊抗小鼠IgG抗体(稀释度1：2000)，每孔100μl。37℃静置1.5h；清洗后加入TMB显色液，一起孵育，加入终止液停止反应；使用酶标仪读取OD450处吸光度。
C)细胞免疫和体液免疫偏向型抗体水平的检测
参照上面测试IgG滴度的方法，采集本发明实施例6步骤A免疫后56d小鼠血清，评估疫苗诱导细胞免疫和体液免疫偏向型抗体水平。
二、实验结果
各组的特异性IgG抗体滴度见图5。图5为实施例4所构建的佐剂组合物疫苗(MnCpG-Ag组和Mn2CpG-Ag组)与本实施例其他组免疫后19、35和56天小鼠血清RBD特异性IgG抗体滴度。
由图5可知，实施例11所构建的佐剂组合物疫苗(MnCpG-Ag组和Mn2CpG-Ag 组)可以在小鼠体内有效增强新冠疫苗引起的抗体反应。通过对多个稀释度血清抗体的检测，可以看出实施例11所构建的佐剂组合物疫苗(MnCpG-Ag组和Mn2CpG-Ag组)可以有效增强IgG免疫反应水平。在载带同等抗原剂量的情况下(均为10微克)，实施例8所构建的佐剂组合物疫苗组(MnCpG-Ag组和Mn2CpG-Ag组)抗体滴度水平是单纯四氧化三锰颗粒佐剂Mn-Ag组疫苗的4倍，是单纯Alum佐剂疫苗抗体的10倍，是单纯CpG佐剂组疫苗抗体的50倍；表明与CpG形成佐剂组合可以提升四氧化三锰颗粒佐剂的免疫增效能力。
从56d的抗体结果可见，本实施例12中的MnCpG-Ag组产生的抗体是Mn2CpG组的2.2倍，表明通过共价修饰结合CpG的纳米颗粒佐剂能产生比吸附方式结合CpG的微米颗粒疫苗所产生的抗体水平更高。另外，Mn2CpG-Ag组抗体水平是Mn-Ag组的2倍，表明即使是吸附方式来制备佐剂组合物产生的抗体也比单纯四氧化三锰佐剂的组高；可见，无论吸附(实施例2)还是共价修饰(实施例1)方案组合CpG和四氧化三锰颗粒佐剂的结合都能显著提升各自的疫苗增效能力，相较于纯抗原Ag组及CpG-Ag组促进更高抗体产生水平。
实施例11构建的佐剂组合物疫苗诱导细胞和体液免疫平衡效果对比见图6。图6为本发明实施例11所构建的佐剂组合物疫苗与实施例12中不同配方疫苗诱导细胞和体液免疫平衡效果评估。由图6可知，实施例11得到的佐剂组合物MnCpG和Mn2CpG制备的疫苗引起的IgG2C/IgG1的比例接近1，诱导了平衡的T细胞反应；而Mn-Ag组IgG2C/IgG1的比例小于1，表明单纯锰佐剂Mn-Ag组诱导Th2偏向型免疫反应，CpG佐剂疫苗诱导Th1偏向型免疫反应。
实施例14佐剂组合物疫苗诱导的中和抗体应答
一、实验方法
实施例12的步骤A得到的疫苗免疫后56天各组小鼠血清样本进行假病毒感染中和试验，具体过程如下：将含有假病毒(50μl；购买自Sino Biological公司，货号:PSV001)的上清液与连续稀释的小鼠血清在37℃下预孵育1小时，然后添加到表达ACE2的293T细胞(5×104细胞)中。24小时后加入新鲜培养基，然后使用市售细胞裂解缓冲液裂解细胞。加入荧光素酶底物后，在发光计(Bio-Tech)中测定相对荧光素酶活性。计算假病毒中和效率，并表示为50％中和抗体滴度。
二、实验结果
结果见图7，图7为实施例11构建的佐剂组合物疫苗与实施例12构建的其他配方疫 苗针对假病毒的体外免疫效果对比。由图7可知，实施例11得到的佐剂组合物MnCpG和Mn2CpG制备的疫苗可诱导显著增强的针对新型冠状病毒的中和抗体应答。但MnCpG-Ag与Mn2CpG-Ag组血清对新冠假病毒中和能力相当。
实施例15佐剂组合物增强流感亚单位疫苗保护效果评估
一、佐剂组合物流感亚单位疫苗制备
将本实施例11步骤A或B制得的佐剂组合物MnCpG和佐剂组合物Mn2CpG分别分散入生理盐水中，制成1mg/ml溶液，并移取100微升溶液，加入5μg流感亚单位抗原(记为H1N1 HA，购自义翘神州，货号40731-V07H)震荡或搅拌混合均匀后，置于旋转式摇床混合10～60分钟，得到基于四氧化三锰颗粒佐剂与CpG佐剂的佐剂组合物载带流感亚单位抗原的流感亚单位疫苗，MnCpG-HA与Mn2CpG-HA。
二、流感亚单位疫苗免疫小鼠抗体水平评估
将制备好的佐剂组合物流感亚单位疫苗MnCpG-HA与Mn2CpG-HA通过肌肉注射进行免疫，测定产生的抗原特异性IgG、IgM抗体滴度。
具体方法如下：
实验动物：Balb/C小鼠，6～8周，5只/组，雌性。
给药剂量：H1N1 HA抗原5μg/鼠；商品铝佐剂Alum(购自Invivogen公司,CAS:21645-51-2)：100μg/100μl/鼠；MnCpG-HA与Mn2CpG-HA：100μg/100μl/鼠；
实验组及给药剂量：(1)HA组：H1N1 HA抗原5μg/鼠；(2)Alum-HA组：商品铝佐剂Alum 100μg/100μl+H1N1 HA 5μg/鼠；(3)MnCpG-HA组：100μl注射液，含MnCpG 100μg和H1N1 HA抗原5μg/鼠；(4)Mn2CpG-HA组：100μl注射液，含Mn2CpG 100μg和H1N1 HA抗原5μg/鼠。
免疫方案：各组疫苗免疫小鼠后，小鼠按照免疫分组在首次免疫后第3周加强免疫，并于二次免疫后2周，眼眶取血，测定血清中抗体滴度。
血清中的抗体滴度采用酶联免疫吸附法(ELISA)检测，评估小鼠血清中疫苗诱导的IgG和IgM水平。
三、实验结果
图8为本申请的实施例11步骤A和步骤B制备的佐剂组合物MnCpG、佐剂组合物Mn2CpG或商品铝佐剂Alum载带流感H1N1 HA抗原对小鼠进行免疫后，产生的特异性抗体滴度分析结果。
实验结果如图8A，本发明实施例11步骤A和步骤B得到的佐剂组合物 MnCpG、佐剂组合物Mn2CpG制备的疫苗相较于商品铝佐剂制备的疫苗更好地增强免疫应答，产生的抗体滴度分别为商业铝佐剂的137倍和96倍。
实验结果如图8B，本发明实施例11步骤A和步骤B得到的佐剂组合物MnCpG、佐剂组合物Mn2CpG制备的疫苗相较于商品铝佐剂制备的疫苗具有更高的IgM水平，产生的抗体滴度分别为铝佐剂的4.3倍和3.1倍。
图8结果表明，本实施例11步骤A和步骤B制备的佐剂组合物MnCpG、佐剂组合物Mn2CpG制备的疫苗相较于商品铝佐剂制备的疫苗能够更加有效的免疫增效能力。
实施例16四氧化三锰纳米颗粒佐剂与其它寡核苷酸组合的佐剂组合物的制备
A)5ml 5mg/ml的实施例3制备的四氧化三锰颗粒的MES缓冲液溶液，pH6左右，与1ml 0.02mmol/ml EDC的pH6左右的MES缓冲液溶液混合，35℃活化反应30分钟后，加入1ml 0.02mmol/ml NHS的pH6左右的MES缓冲液溶液，继续活化2小时，之后透析除去未反应的EDC和NHS以及反应副产物，之后加入2ml 0.04mmol/ml ATP溶液混合，维持pH 8.0条件，在室温300rpm搅拌下反应2小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物(佐剂组合物Mn3ATP)。
B)10ml 10mg/ml的实施例4制备的四氧化三锰颗粒的MES缓冲液溶液，pH 5.7左右，与2ml 0.05mmol/ml EDC的pH 5.7左右的MES缓冲液溶液混合，35℃活化反应30分钟后，加入2ml 0.05mmol/ml NHS的pH 5.7左右的MES缓冲液溶液，继续活化2小时，之后透析除去未反应的EDC和NHS以及反应副产物，之后加入5ml 0.1mmol/ml ADP溶液混合，维持pH 7.8条件，在室温600rpm搅拌下反应15小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物(佐剂组合物Mn4ADP)。
C)5ml 10mg/ml的实施例5制备的四氧化三锰颗粒的MES缓冲液溶液，pH 5.7左右，与2ml 0.05mmol/ml EDC的pH 5.7左右的MES缓冲液溶液混合，35℃活化反应30分钟后，加入2ml 0.05mmol/ml马来酰亚胺的pH 8.7左右的MES缓冲液溶液，继续活化2小时，之后透析除去未反应的EDC和NHS以及反应副产物，之后加入5ml 0.1mmol/ml AMP溶液混合，维持pH 8.7条件，在室温900rpm搅拌下反应24小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与ATP的佐剂组合物(佐剂组合物Mn5AMP)。
D)将5ml 0.2mmol/ml的实施例6制备的四氧化三锰颗粒的水溶液与等体积 0.0005mmol/ml巯基化修饰的CpG(购自生工生物，tcgtcgttttcggcgcgcgccg-SH)混合，维持pH8.4条件，在室温500rpm搅拌下反应2小时后，超滤提纯后，完成四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物的制备，得到四氧化三锰纳米颗粒佐剂与CpG的佐剂组合物(佐剂组合物Mn6CpG)。
实施例17复合疫苗的制备以及增强寄生虫保护效果评估
弓形虫病相关防护研究中新型致密颗粒蛋白GRA是寄生虫在主动侵入宿主细胞时分泌的排泄-分泌抗原(ESAs)的主要成分之一，与寄生虫的细胞内维持有关。相关研究表明可以将其作为疫苗药物。
将实验小鼠进行随机分组，每组10只：
①Ctr组(对照组)：注射液体积为100微升，注射液为生理盐水；
②Ag组：注射液体积为100微升，注射10μg GRA抗原；
③ATP-Ag组：注射液体积为100微升，注射10μg GRA抗原与ATP佐剂复合；
④ADP-Ag组：注射液体积为100微升，注射10μg GRA抗原与ADP佐剂复合；
⑤Mn3-Ag组：注射液体积为100微升，注射载带10μg GRA抗原的含锰元素25μg本发明实施例3制备的四氧化三锰颗粒佐剂构建的纳米疫苗；
⑥Mn4-Ag组：注射液体积为100微升，注射载带10μg RBD抗原的50μg商品铝佐剂Alum(Invivogen公司，
Figure PCTCN2023079905-ftappb-I100005
adjuvant 2％，CAS:21645-51-2)；
⑦Mn3ATP-Ag组：注射液体积为100微升，注射实施例16步骤A所构建的佐剂组合物疫苗，即载带10μg RBD抗原(Ag)的Mn3ATP。
⑧Mn4ADP-Ag组：注射液体积为100微升，注射实施例16步骤B所构建的疫苗，即载带10μg RBD抗原(Ag)的Mn4ADP。
将实施例16制得的佐剂组合物，分别与10微克GRA疫苗药物混合，并已无佐剂组合物组做对比，对实验小鼠进行免疫注射。注射5周后，对各实验组小鼠进行用弓形虫RH株感染。每天观察和记录各试验组种小鼠的存活率，实验结果如下表所示：
表2、各实验组中小鼠存活率随天数变化统计(存活率*100％)
Figure PCTCN2023079905-ftappb-I100006

Figure PCTCN2023079905-ftappb-I100007
由此表可知，实施例3，4所构建的四氧化三锰纳米佐剂以及实施例16所构建的组合纳米佐剂Mn3ATP以及Mn4ADP能够显著增强原有GRA疫苗的免疫效果。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (37)

  1. 四氧化三锰颗粒在制备疫苗佐剂中的应用，其特征在于，所述佐剂为颗粒佐剂，所述颗粒佐剂为外部包裹有或没有赋形剂的四氧化三锰颗粒，所述颗粒佐剂粒径为5～3000nm。
  2. 根据权利要求1所述的应用，其特征在于，所述四氧化三锰颗粒外部包裹有赋形剂，所述赋形剂为蛋白、多肽、聚合物、核酸、多糖中的一种或几种，所述赋形剂能够与单链核苷酸佐剂通过化学选择性共价修饰进行反应。
  3. 根据权利要求2所述的应用，其特征在于，所述单核苷酸佐剂为含有CpG ODN的寡核苷酸。
  4. 根据权利要求2所述的应用，其特征在于，所述化学选择性共价修饰是基于所述赋形剂与所述单链核苷酸佐剂所携带化学选择性共价修饰基团对；所述化学选择性共价修饰包括：马来酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺、琥珀酰亚胺和/或1-(3-二甲氨基丙基)-3-乙基碳二亚胺。
  5. 根据权利要求2所述的应用，其特征在于，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～4000)：1。
  6. 根据权利要求5所述的应用，其特征在于，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～400)：1。
  7. 根据权利要求6所述的应用，其特征在于，四氧化三锰颗粒中锰元素与赋形剂的摩尔比为(20～300)：1。
  8. 根据权利要求2～7任一所述的应用，其特征在于，所述外部包裹有赋形剂的四氧化三锰颗粒制备方法为：二价锰盐水溶液与赋形剂分子溶液充分混合得到混合溶液，再与碱性的溶液混合，充分反应，透析纯化，冻干，得到外部包裹有赋形剂的四氧化三锰颗粒。
  9. 根据权利要求8所述的应用，其特征在于，碱性的溶液混合后，溶液pH值为6～14。
  10. 根据权利要求9所述的应用，其特征在于，碱性的溶液混合后，溶液pH值为6.5～7.4。
  11. 根据权利要求8所述的应用，其特征在于，充分混合的条件为：温度为30～37℃，搅拌反应0.2～5小时。
  12. 根据权利要求8所述的应用，其特征在于，充分反应的条件为：先于温度为30～ 37℃反应0.2～5小时，后升高温度至60～95℃反应0.2～5小时。
  13. 一种佐剂组合物，其特征在于，所述佐剂组合物含有颗粒佐剂和单链核苷酸佐剂，所述颗粒佐剂为权利要求1到7任一中所述的外部包裹有或没有赋形剂的四氧化三锰颗粒。
  14. 根据权利要求13所述的佐剂组合物，其特征在于，所述单链核苷酸佐剂为含有CpG ODN的寡核苷酸。
  15. 根据权利要求14所述的佐剂组合物，其特征在于，所述寡核苷酸为DNA片段、ATP、ADP和/或AMP。
  16. 根据权利要求15所述的佐剂组合物，其特征在于，所述DNA片段为改性DNA片段或未改性DNA片段。
  17. 根据权利要求16所述的佐剂组合物，其特征在于，所述改性DNA片段为对DNA片段进行氨基、羧基或巯基官能团功能化、化学选择性的共价修饰基团功能化，包括马来酰亚胺化、琥珀酰亚胺化、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐功能化修饰。
  18. 根据权利要求17所述的佐剂组合物，其特征在于，所述单链核苷酸佐剂为：tcgtcgttttcggcgcgcgccg-SH。
  19. 一种疫苗佐剂，其特征在于，其制备方法为：将权利要求15所述的佐剂组合物中的颗粒佐剂和单链核苷酸佐剂在pH为6～9的缓冲液中通过非共价吸附或化学选择性共价修饰反应偶联0.5～24小时，经纯化即得。
  20. 根据权利要求19所述的疫苗佐剂，其特征在于，所述颗粒佐剂为外部包裹有赋形剂的四氧化三锰颗粒，颗粒佐剂通过共价修饰与单链核苷酸佐剂偶联；所述颗粒佐剂为外部没有包裹赋形剂的四氧化三锰颗粒，颗粒佐剂通过吸附与单链核苷酸佐剂相结合。
  21. 根据权利要求19所述的疫苗佐剂，其特征在于，所述通过化学选择性共价修饰反应偶联为将所述佐剂组合物中的颗粒佐剂和单链核苷酸佐剂进行功能基团活化，之后利用活化的功能基团将颗粒佐剂和单链核苷酸佐剂通过化学选择性共价修饰反应偶联。
  22. 根据权利要求19所述的疫苗佐剂，其特征在于，通过EDC/NHS反应、静电或配位作用吸附将所述颗粒佐剂和所述单链核苷酸偶联。
  23. 一种疫苗，其特征在于，含有权利要求13所述的佐剂组合物和/或权利要求19所述的疫苗佐剂。
  24. 根据权利要求23所述的疫苗，其特征在于，权利要求13所述的佐剂组合物和/或权利要求19所述的疫苗佐剂载带疫苗抗原。
  25. 根据权利要求24所述的疫苗，其特征在于，所述疫苗抗原包括灭活病原体或者提 取的病原体亚单位抗原、重组亚单位抗原、抗原表位肽、核酸抗原及其组合。
  26. 根据权利要求25所述的疫苗，其特征在于，所述病原体包括病毒、细菌和/或寄生虫。
  27. 根据权利要求26所述的疫苗，其特征在于，所述病原体包括病毒和/或寄生虫。
  28. 根据权利要求27所述的疫苗，其特征在于，所述病毒选自为DNA病毒或RNA病毒；所述病毒选自：冠状病毒科、疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科；所述病毒选自：新型冠状病毒、流感病毒、单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV中的一种或几种。
  29. 根据权利要求28所述的疫苗，其特征在于，所述病毒选自：新型冠状病毒和/或流感病毒。
  30. 根据权利要求27所述的疫苗，其特征在于，所述寄生虫选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫中的一种或几种。
  31. 一种疫苗的制备方法，其特征在于，权利要求13所述的佐剂组合物和/或权利要求19所述的疫苗佐剂与疫苗抗原充分混合，即得。
  32. 根据权利要求31所述的制备方法，其特征在于，所述疫苗抗原包括灭活病原体或者提取的病原体亚单位抗原、重组亚单位抗原、抗原表位肽、核酸抗原及其组合。
  33. 根据权利要求32所述的制备方法，其特征在于，所述病原体包括病毒、细菌和/或寄生虫。
  34. 根据权利要求33所述的制备方法，其特征在于，所述病原体包括病毒和/或寄生虫。
  35. 根据权利要求34所述的制备方法，其特征在于，所述病毒选自为DNA病毒或RNA病毒；所述病毒选自：冠状病毒科、疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科；所述病毒选自：新型冠状病毒、流感病毒、单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV中的一种或几种。
  36. 根据权利要求35所述的制备方法，其特征在于，所述病毒选自：新型冠状病毒和/或流感病毒。
  37. 根据权利要求34所述的制备方法，其特征在于，所述寄生虫选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫中的一种或几种。
PCT/CN2023/079905 2022-01-07 2023-03-06 四氧化三锰颗粒在制备疫苗佐剂中的应用 WO2023131356A2 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2023205611A AU2023205611A1 (en) 2022-01-07 2023-03-06 Use of trimanganese tetraoxide particles in preparation of vaccine adjuvant
IL314123A IL314123A (en) 2022-01-07 2023-03-06 Use of trimanganese tetroxide particles to prepare a material that helps to assemble

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210018495.5 2022-01-07
CN202210018495.5A CN114432437A (zh) 2022-01-07 2022-01-07 四氧化三锰颗粒在制备疫苗佐剂中的应用

Publications (2)

Publication Number Publication Date
WO2023131356A2 true WO2023131356A2 (zh) 2023-07-13
WO2023131356A3 WO2023131356A3 (zh) 2023-08-24

Family

ID=81368364

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/079905 WO2023131356A2 (zh) 2022-01-07 2023-03-06 四氧化三锰颗粒在制备疫苗佐剂中的应用

Country Status (4)

Country Link
CN (1) CN114432437A (zh)
AU (1) AU2023205611A1 (zh)
IL (1) IL314123A (zh)
WO (1) WO2023131356A2 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114432437A (zh) * 2022-01-07 2022-05-06 广东粤港澳大湾区国家纳米科技创新研究院 四氧化三锰颗粒在制备疫苗佐剂中的应用
CN114848810B (zh) * 2022-05-11 2023-10-10 江南大学 一种手性纳米疫苗及其制备方法与应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2008200021A1 (en) * 2001-08-17 2008-01-31 Coley Pharmaceutical Gmbh Combination motif immune stimulatory oligonucleotides with improved activity
CN107456575A (zh) * 2017-09-06 2017-12-12 苏州大学 一种二氧化锰纳米佐剂及其制备方法、应用
CN108714213A (zh) * 2018-06-04 2018-10-30 北京工业大学 一种自组装的纳米佐剂及由该佐剂形成的纳米疫苗的制备方法与应用
KR20230005814A (ko) * 2020-04-06 2023-01-10 발네바 오스트리아 게엠베하 Cpg-어쥬번트된 sars-cov-2 바이러스 백신
CN112791181B (zh) * 2021-02-05 2024-04-02 广东粤港澳大湾区国家纳米科技创新研究院 一种锰纳米佐剂、其制备方法及用途
CN113274492B (zh) * 2021-05-21 2022-06-17 大连理工大学 一种基于羟基氧化铝纳米羧基改性的复合疫苗佐剂的制备方法
CN113797329A (zh) * 2021-10-19 2021-12-17 启锰生物科技(江苏)有限公司 一种二价锰佐剂和CpG佐剂的疫苗佐剂组合物及其制作方法
CN114432437A (zh) * 2022-01-07 2022-05-06 广东粤港澳大湾区国家纳米科技创新研究院 四氧化三锰颗粒在制备疫苗佐剂中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMERG MICROBES INFECT, vol. 10, no. 1, 2021, pages 1555 - 1573
no. 21645-51-2

Also Published As

Publication number Publication date
IL314123A (en) 2024-09-01
WO2023131356A3 (zh) 2023-08-24
AU2023205611A1 (en) 2024-08-22
CN114432437A (zh) 2022-05-06

Similar Documents

Publication Publication Date Title
WO2023131356A2 (zh) 四氧化三锰颗粒在制备疫苗佐剂中的应用
Sayın et al. Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan (TMC) nanoparticles for non-invasive vaccine delivery
Sayın et al. TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines
Zhao et al. Enhancing mucosal immune response of Newcastle disease virus DNA vaccine using N-2-hydroxypropyl trimethylammonium chloride chitosan and N, O-carboxymethyl chitosan nanoparticles as delivery carrier
US8961983B2 (en) Mucosal vaccine using cationic nanogel
CN111658780B (zh) 一种基于阴离子聚合物及其衍生物制备的疫苗载体
WO2023134783A2 (zh) 一种铝纳米晶递送系统及其结合疫苗抗原分子自组装颗粒佐剂疫苗
WO2022165980A1 (zh) 一种锰纳米佐剂、其制备方法及用途
Nanda et al. Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1–OmpA in guinea pigs
CN108434089A (zh) 使用淀粉的口服疫苗快速溶解剂型
CN109701010B (zh) 疫苗复合佐剂系统及其在抗原中的应用
CN1404399A (zh) 调节包括与粘膜体表面接触的疫苗抗原的物质的作用的新的非抗原性粘膜佐剂制剂
CN103002908B (zh) 粘膜疫苗
Zhao et al. Synthesis, characterization, and immune efficacy of layered double hydroxide@ SiO2 nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine
CN101443039A (zh) 用cd1d配体进行免疫的组合物和方法
He et al. Cistanche deserticola polysaccharide-functionalized dendritic fibrous nano-silica as oral vaccine adjuvant delivery enhancing both the mucosal and systemic immunity
CN112641935A (zh) 一种含颗粒载体的亚单位疫苗及其应用
CN107050446B (zh) 经修饰的季节流感-rsv联合疫苗及其制备方法
Kumar et al. Exploration of chitosan and its modified derivatives as vaccine adjuvant: a review
CN104740625A (zh) 负载新城疫病毒F基因质粒DNA的LDH@SiO2纳米粒的制备方法
Chadhar et al. Development and characterization of tetanus toxoid loaded trimethylchitosan chloride nanoparticles for nasal immunization
CN104189922B (zh) 一种载新城疫DNA疫苗Ag@SiO2纳米粒的制备方法
WO2016093358A1 (ja) ロッド型ナノ粒子及び核酸からなる組成物
CN109701011B (zh) 疫苗复合佐剂系统及其在抗原中的应用
KR102643430B1 (ko) pH 감응형 나노 복합체를 활용한 백신 전달체

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23737218

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 314123

Country of ref document: IL

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112024013862

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: AU23205611

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2023737218

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023737218

Country of ref document: EP

Effective date: 20240807

ENP Entry into the national phase

Ref document number: 2023205611

Country of ref document: AU

Date of ref document: 20230306

Kind code of ref document: A