WO2022165980A1 - 一种锰纳米佐剂、其制备方法及用途 - Google Patents

一种锰纳米佐剂、其制备方法及用途 Download PDF

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WO2022165980A1
WO2022165980A1 PCT/CN2021/085578 CN2021085578W WO2022165980A1 WO 2022165980 A1 WO2022165980 A1 WO 2022165980A1 CN 2021085578 W CN2021085578 W CN 2021085578W WO 2022165980 A1 WO2022165980 A1 WO 2022165980A1
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manganese
adjuvant
nano
template
antigen
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French (fr)
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陈春英
王亚玲
刘野
郭梦雨
陈曦
胡栩豪
赵宇亮
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广东粤港澳大湾区国家纳米科技创新研究院
国家纳米科学中心
中国医学科学院医学生物学研究所
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biomedical technology and vaccine technology, in particular to a manganese nano-adjuvant, a preparation method and use thereof.
  • Aluminum adjuvant is recognized as a safe adjuvant. Compared with free antigen, it can promote antigen to generate better immune response, but at the same time, lack of cellular immunity is a natural defect of alum vaccine.
  • LN lymph node
  • B cells immune cells
  • the size-restricting nature of lymph nodes makes it difficult to deliver vaccines exclusively to immune cells.
  • the unique size and surface properties of functional nanomaterials help deliver vaccine components (antigens and adjuvants) to critical immune cells or lymphoid tissues and improve immune responses to prevent infection.
  • albumin can confer targeting capabilities to vaccines, eg, the delivery of adjuvants such as Evans blue or lipo-CpG to lymph nodes, thereby facilitating induction of an effective immune response.
  • albumin is also a good template for biomineralization to prepare inorganic nanoparticles.
  • Manganese adjuvant is a new type of adjuvant developed in recent years. At present, there have been reports on the application of divalent manganese and tetravalent manganese as vaccine adjuvants.
  • publication number CN107412260A discloses the use of divalent manganese in the preparation of a medicine for improving innate immunity or/or adaptive immunity.
  • publication number is CN111821316A, which discloses a manganese composition containing divalent manganese for immune enhancement.
  • publication number CN107456575A discloses a manganese dioxide nano-adjuvant and its preparation method and application.
  • the immune enhancement effect of simple manganese adjuvant or tetravalent manganese adjuvant needs to be improved.
  • the present invention provides a manganese nano-adjuvant, its preparation method and use.
  • the manganese nano-adjuvant provided by the invention can effectively carry the immune antigen, and can obtain more excellent immunotherapy effect with less antigen loading and lower injection dosage; and can effectively deliver the immune antigen to the lymph nodes tissue, and greatly enhance the cellular internalization of immune antigens, and highly efficient activation of immune cells.
  • the invention provides the application of trimanganese tetroxide particles in the preparation of manganese nano-adjuvant.
  • the manganese nano-adjuvant comprises trimanganese tetroxide nanoparticles and template molecules wrapped around them, and the molar ratio of template molecule to manganese element is 1 : (10 ⁇ 1000), the template molecule includes template protein and its fragments or polypeptides.
  • the molar ratio of template molecule to manganese element is 1:(200-400).
  • the template protein includes one or more of bovine serum albumin, human serum albumin, mouse serum albumin, transferrin, and antigenic proteins; the polypeptides include the above-mentioned template protein-derived fragments and antigenic polypeptides.
  • the average particle size of the manganese nanoadjuvant is 1-100 nm.
  • the invention also provides a preparation method of a manganese nano-adjuvant, comprising the following steps:
  • the template molecules include template proteins and fragments or polypeptides thereof.
  • the divalent manganese salt is selected from one or more of manganese chloride, manganese nitrate, manganese acetate, and manganese sulfate; but the present invention is not limited to this, and the divalent manganese salt types recognized by those skilled in the art are all in within the protection scope of the present invention.
  • the reagent for adjusting the pH value of the composite solution is an alkaline reagent
  • the alkaline reagent is sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia water, triethylamine, pyridine, N-methylmorpholine, tetramethylethyl acetate one or more of the diamines;
  • the concentration of the divalent manganese salt is 0.1-0.5 mol/L, and the concentration of the template protein is 1-50 g/L.
  • the stirring temperature is 30-37° C.
  • the stirring time is 1-10 hours
  • the stirring speed is 200-1000 r/min.
  • the present invention also provides the manganese nano-adjuvant prepared by the above method.
  • the present invention also provides the application of the manganese nano-adjuvant in preparing a vaccine, and the vaccine includes the above-mentioned manganese nano-adjuvant.
  • the present invention also provides a vaccine, comprising antigen and the above-mentioned manganese nano-adjuvant, and the mass ratio of manganese element in the antigen and manganese nano-adjuvant is 1:(0.025-40).
  • the mass ratio of the manganese element in the antigen and the manganese nano-adjuvant is 1:(0.1-10).
  • the antigen is a protein antigen and/or a polypeptide antigen.
  • the antigen is one or more of novel coronavirus antigen, HIV antigen, Mycobacterium tuberculosis antigen, malaria antigen, human papilloma virus antigen or tumor-associated antigen.
  • the present invention also provides a method for preparing the above vaccine, wherein the manganese nano-adjuvant and the antigen are incubated in a buffer solution at 20-37° C. for 30-120 minutes.
  • the buffer is one of PBS buffer, Tris-HCl buffer or citrate (salt) buffer.
  • the invention provides a manganese nano-adjuvant, its preparation method and use.
  • the manganese nano-adjuvant includes manganese tetroxide nanoparticles and a template molecule wrapped outside, the molar ratio of template molecule and manganese element is 1:(10-1000), and the template molecule includes template protein and its fragments or polypeptides.
  • the technical effect that the present invention has is:
  • the invention provides a preparation method of manganese nano-adjuvant based on manganese tetroxide nanoparticles prepared by protein mineralization and a strategy for constructing a nano-vaccine drug based on the manganese nano-adjuvant, which can effectively prevent virus infection.
  • the provided manganese nanoadjuvant is a programmable platform, which is easy to synthesize and can efficiently enhance the lymph node delivery efficiency of antigen molecules as well as antigen immunogenicity.
  • the manganese nano-adjuvant in the constructed nano-vaccine can stably and effectively carry antigen molecules, and can effectively enhance the response of antigen-specific antibodies and T cells.
  • the natural targeting properties of albumin and the size effect of nanovaccine make this manganese nanoadjuvant suitable for efficient drainage and retention in lymph nodes, greatly increasing cellular uptake and activation of immune cells, thereby enhancing antigen-specific immune responses.
  • the nanovaccine drug constructed with manganese nanoadjuvant can efficiently induce humoral and cellular immune responses in mice even with reduced antigen dose and injection times. More importantly, the high-efficiency induced neutralizing antibody response of the constructed nano-vaccine drug also shows that it has the potential to provide satisfactory protective immunity against the new coronavirus and other types of viruses. Finally, each component of the nano-vaccine drug has good biological adaptability and low cost, which greatly increases the possibility of further clinical research.
  • the preparation process requires high temperature and complicated process, which is not conducive to clinical transformation and application as vaccine adjuvant.
  • the preparation method of the nano-adjuvant of the present invention is simple.
  • Fig. 1 is the preparation flow chart of the manganese nano-adjuvant of the embodiment of the present invention 1;
  • Fig. 2 is the electron transmission microscope image and X-ray diffraction spectrum of the manganese nano-adjuvant prepared in Example 1 of the present invention
  • Fig. 3 is the TEM spectrum of the manganese nano-adjuvant prepared in Example 2 of the present invention.
  • Fig. 4 is the biological toxicity of the manganese nano-adjuvant (MnARK) obtained in Example 1, the cell internalization and in vitro DC maturation promotion results;
  • Figure 5 is a schematic diagram of the preparation of manganese nano-adjuvant and the construction of nano-vaccine drugs
  • Fig. 6 is the manganese nano-adjuvant obtained in Example 1 to construct the nano-vaccine drug data map
  • Figure 7 shows the in vivo accumulation of nano-vaccine drugs and the targeted fluorescence imaging of lymph nodes
  • Fig. 8 is that the nano-vaccine drug constructed in Example 4 activates B cells
  • Fig. 9 is the nano-vaccine medicine that embodiment 4 constructs to induce immunity effect comparison
  • Figure 10 is a comparison of the in vitro immune effects of the nano-vaccine drugs constructed in Example 4.
  • Figure 11 is a comparison of the neutralizing antibody production effect between the nanovaccine constructed in Example 4 and manganese chloride.
  • the invention discloses a manganese nano-adjuvant, a preparation method and application thereof, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications will be apparent to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the manganese nano-adjuvant of the present invention can be purchased from the market.
  • A) Add 5 mL of manganese chloride aqueous solution with a concentration of 0.1 mol/L to 10 mL of bovine serum albumin (BSA) solution with a concentration of 10 mg/mL, and stir and mix to obtain a composite solution.
  • BSA bovine serum albumin
  • step B) Add 0.1 mL of NaOH aqueous solution with a concentration of 2 mol/L to the composite solution in step A, continue stirring at 34° C. and 900 rpm for 2 hours, and obtain a manganese nano-adjuvant (MnARK) product after dialysis, purification and freeze-drying.
  • MnARK manganese nano-adjuvant
  • FIG. 2 is an electron transmission microscope image and an X-ray diffraction spectrum of the manganese nanoadjuvant prepared in Example 1 of the present invention.
  • the average particle size of the manganese nano-adjuvant prepared in Example 1 of the present invention is 9.77 nanometers, and the dispersibility is good and the size is uniform;
  • XRD data shows that the manganese nano-adjuvant prepared in Example 1 of the present invention is a tetragonal crystal phase four. Trimanganese oxide structure.
  • step B) Add 0.5 mL of 0.1 mol/L NaOH aqueous solution to the composite solution of step A, continue to stir at 30° C. 700 rpm for 5 hours, and obtain a manganese nano-adjuvant product after dialysis, purification and freeze-drying.
  • Figure 3 is a TEM image of the manganese nanoadjuvant prepared in Example 2 of the present invention.
  • the average particle size of the manganese nanoadjuvant prepared in Example 2 of the present invention is 28.43 nanometers, with good dispersibility and uniform size.
  • Viability detection experiment DC2.4 cells were seeded in 96-well plates and incubated with different concentrations (0 to 0.5 mmol/L) of manganese chloride or MnARK for 24 hours. Cell viability was assessed using the CCK-8 kit.
  • Figure 4 shows the results of experiments on the biological toxicity of the manganese nanoadjuvant (MnARK) obtained in Example 1, the internalization of the nanoadjuvant by DC2.4 cells and the in vitro promotion of DC maturation.
  • MnARK manganese nanoadjuvant
  • MnARK nanoparticles had no obvious cytotoxicity to DC cells, even at a concentration of 0.5 mmol/L, compared with free manganese ions (manganese chloride). Meanwhile, MnARK can also induce higher levels of DC cell maturation.
  • Example 1 for novel coronavirus, the manganese nano-adjuvant obtained in Example 1 is used in nano-vaccine drug construction and immunotherapy.
  • A) Select the new coronavirus antigen RBD as the immune antigen research object.
  • the fluorescently labeled antigen and the manganese nanoadjuvant obtained in Example 1 were added to PBS, and incubated at 25°C for 60 minutes.
  • the mass ratio of the added antigen molecule and the manganese element in the manganese nano-adjuvant is 1:5.
  • step B Centrifuge the solution obtained in step A at a speed of 10,000g for 20 minutes, and collect the centrifuged precipitate, which is the nanovaccine drug constructed by manganese nanoadjuvant combined with antigen. The concentration of unbound antigen in the supernatant was then measured by fluorescence. Fluorescence measurements were performed using a PerkinElmer fluorescence plate reader.
  • Figure 6 is the obtained manganese nanoadjuvant in Example 1, the adsorption data of antigen molecules at different concentrations; the real-time measurement curve of the association and dissociation of the interaction between RBD antigen and manganese nanoadjuvant; and RBD and manganese nanoadjuvant ( Changes in hydrated particle size after MnARK) binding.
  • the nanoprobes obtained in Example 1 can effectively adsorb and carry antigen molecules; RBD antigens are stably combined with manganese nanoadjuvants with high affinity; the nano-vaccine drug hydrated particle size formed after combining with RBD antigens Compared with the manganese nanoadjuvant, the increase was obvious, indicating that the RBD antigen and the manganese nanoadjuvant achieved an effective combination.
  • mice under the premise of following the national animal health protocol, BALB/c mice aged 6-8 weeks were selected and divided into 3 groups, 12 mice in each group, and the following reagents were injected into the thigh muscle of the right hind leg.
  • the nano-vaccine drug constructed with 25 ⁇ g of manganese nanoadjuvant with the RBD antigen modified by the molecule Cy5; 2, the RBD antigen modified by the fluorescent molecule Cy5 was loaded; 3, the normal saline.
  • mice Fluorescence imaging analysis of mice at injection time points of 0 hours, and 12, 24, 48, and 72 hours. After 12, 24, 48, and 72 hours of fluorescence imaging, 3 mice were collected to collect axillary and inguinal lymph nodes for fluorescence imaging analysis.
  • Figure 7 shows the in vivo accumulation of nanovaccine drugs and the targeted fluorescence imaging of lymph nodes.
  • the nanovaccine drug constructed with manganese nanoadjuvant induced greater accumulation of RBD antigen at the injection site, and the antigen persisted at the site for more than 3 days. Meanwhile, the nanovaccine drug elicited stronger fluorescent signals in the lymph nodes. Quantitative analysis showed that the accumulation efficiency of nanovaccine drugs in lymph nodes was about 2 times higher than that of free RBD antigens at 12h after injection, indicating that the nanovaccine drugs constructed with manganese nanoadjuvant promoted the delivery and effective accumulation of antigens to lymph nodes in vivo.
  • Example 4 The nano-vaccine drug obtained in Example 4 was subjected to ELISPOT analysis using a commercial kit of R&D system (a kit for mouse IFN- ⁇ enzyme-linked immunosorbent spot (ELISPOT) device).
  • R&D system a kit for mouse IFN- ⁇ enzyme-linked immunosorbent spot (ELISPOT) device.
  • the specific process is as follows, the cytokine capture antibody against mouse IFN- ⁇ (diluted 200 times with sterile PBS) was coated on polyvinylidene fluoride membrane (PVDF) in a 96-well plate and incubated at 4°C overnight. 96-well plates were blocked with complete 1640 medium containing 10% fetal bovine serum for 2 hours at room temperature.
  • PVDF polyvinylidene fluoride membrane
  • Fig. 8 shows that the nanovaccine drug constructed in Example 4 activates B cells.
  • mice aged 6-8 weeks were selected for inoculation 3 times.
  • the number of mice in each group was 6.
  • the first mouse thigh intramuscular injection was taken as day 0, the second inoculation was on day 21, and the third inoculation was on day 42, and serum samples were collected on day 57.
  • mice aged 6-8 weeks were selected for inoculation.
  • the number of mice in each group was 6.
  • the nano-vaccine drug constructed with 25 ⁇ g of manganese nano-adjuvant was inoculated twice; 2, 175 ⁇ g of aluminum adjuvant (purchased by Invivogen Company) carrying 50 ⁇ g RBD antigen was inoculated three times; 3, 50 ⁇ g RBD antigen, without nano-adjuvant , inoculated three times.
  • the first mouse thigh intramuscular injection was taken as day 0, the second inoculation was on day 21, and the third inoculation was on day 42, and serum samples were collected on day 57.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Figure 9 is a comparison of the immunity-inducing effects of the nano-vaccine drugs constructed in Example 4.
  • the nanovaccine drug obtained in Example 4 can be more effective than RBD alone (50 ⁇ g) or commercial aluminum adjuvant-loaded RBD even under the condition of lower immune antigen loading (10 ⁇ g). (50 ⁇ g), induced about 5 times the response intensity of IgG and IgM; and under the same antigen loading conditions, two injections of the nanovaccine drug constructed in Example 4 can be compared with three injections of single RBD or commercial aluminum adjuvant. The RBD-induced IgG signal of the band was increased by 10-fold and the IgM signal was increased by 5-fold. These results indicate that the nanovaccine drug constructed in Example 4 can generate stronger immune response after receiving less antigen injection amount (antigen loading amount and/or injection times).
  • step A of Example 7 The four groups of mouse serum samples obtained in step A of Example 7 were subjected to pseudovirus infection neutralization test, and the specific process was as follows: the supernatant containing pseudovirus (50 ⁇ l; purchased from Sino Biological Company) was mixed with serially diluted small Murine serum was pre-incubated for 1 hour at 37°C and then added to ACE2 expressing 293T cells ( 5 x 104 cells). Fresh medium was added after 24 hours and cells were lysed using commercially available cell lysis buffer. After addition of luciferase substrate, relative luciferase activity was determined in a luminometer (Bio-Tech). Pseudovirus neutralization efficiencies were calculated and expressed as 50% and 90% neutralizing antibody titers.
  • mice serum samples obtained in step A of Example 7 were subjected to a new coronavirus live virus infection neutralization test, specifically as follows: the mouse serum was mixed with live virus after 2-fold gradient dilution, and the temperature was 37° C. Incubate for 1 h and add to ACE2-expressing 293T cells in triplicate. The cytopathic effect (CPE) of each well was observed daily and recorded one week after infection. Neutralizing titers of mouse antisera that fully prevented CPE were calculated.
  • CPE cytopathic effect
  • Figure 10 is a comparison of the in vitro immune effects of the nano-vaccine drug constructed in Example 4 against pseudovirus and novel coronavirus live virus.
  • nanovaccine drug obtained in Example 4 can still induce significantly enhanced neutralizing antibody responses against the novel coronavirus even with relatively low antibody loading.
  • step B) The vaccine obtained in step B), under the premise of following the national animal health protocol, select 6-8 week-old BALB/c mice to inoculate 3 times, the number of mice in each group is 6, and there are five groups in total, Respectively, 1, a nano-vaccine drug constructed with 25 ⁇ g of manganese nanoadjuvant loaded with 10 ⁇ g RBD antigen; 2, a vaccine constructed with 25 ⁇ g of Mn@HA adjuvant loaded with 10 ⁇ g RBD antigen; 3, constructed with 25 ⁇ g of manganese chloride with 10 ⁇ g RBD antigen vaccine; 4, 10 ⁇ g RBD antigen, without nano-adjuvant; 5, normal saline group.
  • the first mouse thigh intramuscular injection was taken as day 0, the second inoculation was on day 21, the third inoculation was on day 42, and serum samples were collected on day 57.
  • the IgG levels in the sera of the mice induced by the vaccine in Step A and Step B were assessed by traditional enzyme-linked immunosorbent assay (ELISA).

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Abstract

一种锰纳米佐剂、其制备方法及在制备疫苗中的用途，该锰纳米佐剂包括四氧化三锰纳米颗粒和包裹于其外的模板分子，模板分子与锰元素的摩尔比为1：(10～1000)，模板分子包括模板蛋白及其片段或多肽，模板蛋白选自牛血清白蛋白、人血清白蛋白、鼠血清白蛋白、转铁蛋白、抗原蛋白中的一种或几种，该锰纳米佐剂的平均粒径为1-100nm。该锰纳米佐剂能够有效将免疫抗原递送至淋巴结组织，并增强免疫抗原的细胞内在化，和高效激活免疫细胞。

Description

一种锰纳米佐剂、其制备方法及用途
本申请要求于2021年02月05日提交中国专利局、申请号为202110160471.9、发明名称为“一种锰纳米佐剂、其制备方法及用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药技术与疫苗技术领域，特别涉及一种锰纳米佐剂、其制备方法及用途。
背景技术
虽然现在多种新型冠状病毒疫苗已不断被研发出来，并已被推广到临床前或临床试验中，但是与已经感染人数相比，仍有巨大缺口。除了优化免疫抗原外，使用合适纳米佐剂提升新冠抗原的免疫原性，减少疫苗接种次数和抗原剂量并诱导有效的中和抗体和细胞介导的免疫应答是一种有效阻止新冠继续大流行的潜在应对策略之一。
铝佐剂是公认的安全佐剂，与游离抗原相比，能促进抗原产生更好的免疫反应，但是同时缺乏细胞免疫是明矾配制疫苗的天然缺陷。目前，实现令人满意的抗原递送(靶向淋巴结(LN)和有效的细胞膜通透性)和激活免疫细胞(树突状细胞和B细胞)的疫苗系统的设计和构建仍然是基于蛋白质亚基疫苗所面临的主要问题。淋巴结的尺寸限制特性使得将疫苗专门递送至免疫细胞十分困难。功能性纳米材料的独特尺寸和表面性质有助于将疫苗成分(抗原和佐剂)输送至关键的免疫细胞或淋巴组织，并改善免疫反应以预防感染。
针对以上疫苗发展所面临的困境，设计一种新型的、普适的、可以同时实现对淋巴结的高效传递抗原的纳米佐剂，激活适应性免疫和先天免疫，是一种有效手段。白蛋白的使用可以赋予疫苗靶向能力，例如，将佐剂如伊文思蓝或脂-CpG递送至淋巴结，从而促进诱导有效的免疫应答。同时，白蛋白也是生物矿化制备无机纳米颗粒的良好模板。
锰佐剂是近几年发展起来的新型佐剂。目前，已有关于将二价锰和四价锰应用于疫苗佐剂的报道。如公开号为CN107412260A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。又如公开号为CN111821316A公开了一种用于免疫增强的、包含二价锰的锰组合物。如公开号为CN107456575A公开了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。
发明内容
有鉴于此，本发明提供了一种锰纳米佐剂、其制备方法及用途。本发明所提供的锰纳米佐剂能够有效载带免疫抗原，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，并极大增强免疫抗原的细胞内在化，及极高效的激活免疫细胞。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种四氧化三锰颗粒在制备锰纳米佐剂中应用，锰纳米佐剂包括四氧化三锰纳米颗粒和包裹于其外的模板分子，模板分子与锰元素的摩尔比为1：(10～1000)，模板分子包括模板蛋白及其片段或多肽。
作为优选，锰纳米佐剂中，模板分子与锰元素的摩尔比为1:(200～400)。
作为优选，模板蛋白包括牛血清白蛋白、人血清白蛋白、鼠血清白蛋白、转铁蛋白、抗原蛋白中的一种或几种；多肽包括上述模板蛋白衍生片段、抗原多肽。
作为优选，锰纳米佐剂的平均粒径为1～100nm。
本发明还提供了一种锰纳米佐剂的制备方法，包括如下步骤：
(1)将二价锰盐水溶液与模板分子溶液进行混合，得到复合溶液；
(2)调节复合溶液pH值至9或9以上，搅拌，透析纯化，冻干，得到锰纳米佐剂；
所述模板分子包括模板蛋白及其片段或多肽。
作为优选，二价锰盐选自氯化锰、硝酸锰、乙酸锰、硫酸锰中的一种或几种；但本发明并非限定于此，本领域技术人员认可的二价锰盐种类均在本发明保护范围之内。
作为优选，调节复合溶液pH值的试剂为碱性试剂，碱性试剂为氢氧化钠、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；
作为优选，复合溶液中，二价锰盐的浓度为0.1～0.5mol/L，模板蛋白的浓度为1～50g/L。
作为优选，搅拌的温度为30～37℃，搅拌的时间为1～10小时，搅拌的转速为200～1000r/min。
本发明还提供了由上述方法制备的锰纳米佐剂。
本发明还提供了该锰纳米佐剂在制备疫苗中的应用，疫苗包括上述锰纳米佐剂。
本发明还提供了一种疫苗，包括抗原和上述锰纳米佐剂，抗原与锰纳米佐剂中锰元素的质量比例为1：(0.025～40)。
作为优选，抗原与锰纳米佐剂中锰元素的质量比例为1：(0.1～10)。
作为优选，抗原为蛋白质抗原和/或多肽抗原。
作为优选，抗原为新型冠状病毒抗原、艾滋病毒抗原、结核杆菌抗原、疟疾抗原、人乳头状瘤病毒抗原或肿瘤相关抗原中的一种或多种。
本发明还提供了上述疫苗的制备方法，将锰纳米佐剂与抗原在20～37℃的缓冲液中孵育30～120分钟。
作为优选，缓冲液为PBS缓冲液、Tris-HCl缓冲液或柠檬酸(盐)缓冲液中的一种。
本发明提供了一种锰纳米佐剂、其制备方法及用途。该锰纳米佐剂包括四氧化三锰纳米颗粒和包裹于其外的模板分子，模板分子与锰元素的摩尔比为1：(10～1000)，模板分子包括模板蛋白及其片段或多肽。本发明具有的技术效果为：
本发明提供了一种基于蛋白矿化制备的四氧化三锰纳米颗粒的锰纳米佐剂的制备方法以及基于锰纳米佐剂构建纳米疫苗药物的策略，可以有效预防病毒感染。所提供的锰纳米佐剂是一个可编程平台，合成简便，能够高效增强抗原分子淋巴结递送效率以及抗原免疫原性。所构建的纳米疫苗中锰纳米佐剂能稳定有效地载带抗原分子，高效增强抗原特异性抗体和T细胞反应。白蛋白的天然靶向性质和纳米疫苗的尺寸效应，使该锰纳米佐剂适合于淋巴结中的有效引流和保留，极大增加细胞的摄取和免疫细胞的活化，从而增强抗原特异性免 疫反应。
实验结果表明即使在减少抗原剂量和注射次数的情况下，在小鼠中使用锰纳米佐剂所构建的纳米疫苗药物也能高效引起体液和细胞免疫反应。更重要的是，所构建的纳米疫苗药物高效诱导的中和抗体反应，也表明其具有针对新型冠状病毒以及其他种类病毒能够提供令人满意的保护性免疫的潜力。最后，构建纳米疫苗药物的各个成分生物适应性好，且成本较低，大大增加了下一步临床研究的可能性。
现有无机四氧化三锰纳米粒制备方法多为热分解法、高温水/溶剂热法、油乳法等，制备过程需要高温，流程复杂，不利于作为疫苗佐剂的临床转化应用。本发明纳米佐剂的制备方法简易。
附图说明
图1为本发明实施例1锰纳米佐剂制备流程图；
图2为本发明实施例1制备的锰纳米佐剂的电子透射显微镜图像以及X射线衍射谱图；
图3为本发明实施例2制备的锰纳米佐剂的TEM图谱；
图4为实施例1得到的锰纳米佐剂(MnARK)的生物毒性、细胞内在化以及在体外促进DC成熟实验结果；
图5为锰纳米佐剂制备及纳米疫苗药物构建示意图；
图6为实施例1所得到的锰纳米佐剂构建纳米疫苗药物数据图谱；
图7为纳米疫苗药物活体蓄积及淋巴结靶向荧光成像；
图8为实施例4构建的纳米疫苗药物激活B细胞；
图9为实施例4构建的纳米疫苗药物诱导免疫力效果对比；
图10为实施例4构建的纳米疫苗药物体外免疫效果对比；
图11为实施例4构建的纳米疫苗与氯化锰的中和抗体产生效果对比。
具体实施方式
本发明公开了一种锰纳米佐剂、其制备方法及用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换 和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的锰纳米佐剂、其制备方法及用途中所用原料或试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
制备锰纳米佐剂的步骤如下(图1)：
A)将5mL浓度为0.1mol/L的氯化锰水溶液加入到10mL浓度为10mg/mL的牛血清白蛋白(BSA)溶液中，搅拌混匀得到复合溶液。
B)于步骤A的复合溶液中加入0.1mL浓度为2mol/L的NaOH水溶液，于34℃，900rpm继续搅拌2小时，透析纯化冻干后得到锰纳米佐剂(MnARK)产品。
参见图2。图2为本发明实施例1制备的锰纳米佐剂的电子透射显微镜图像以及X射线衍射谱图。
由图2可知，本发明实施例1制备的锰纳米佐剂平均粒径是9.77纳米，且分散性好、尺寸均一；XRD数据显示本发明实施例1制备的锰纳米佐剂为四方晶相四氧化三锰结构。
实施例2
制备锰纳米佐剂的步骤如下：
A)将10mL浓度为0.2mol/L的硝酸锰水溶液加入到5mL 50mg/mL的人血清白蛋白(HSA)溶液中，搅拌混匀得到复合溶液。
B)于步骤A的复合溶液中加入0.5mL 0.1mol/L的NaOH水溶液，于30℃700rpm继续搅拌5小时，透析纯化冻干后得到锰纳米佐剂产品。
参见图3。图3为本发明实施例2制备的锰纳米佐剂的TEM图像。
由图3可知，本发明实施例2制备的锰纳米佐剂平均粒径是28.43纳米， 且分散性好、尺寸均一。
实施例3
实施例1得到的锰纳米佐剂(MnARK)的生物毒性以及激活免疫细胞活性试验。
A)活力检测实验：将DC2.4细胞接种在96孔板中，分别与不同浓度(0至0.5mmol/L)的氯化锰或MnARK孵育24小时。使用CCK-8试剂盒评估细胞活力。
B)流式细胞分析实验：将DC2.4细胞以每孔3×10 5个细胞的密度接种在6孔板中，并培养12小时，然后用氯化锰或MnARK处理细胞，并孵育24小时后，收集细胞并用抗CD11c、抗CD80和抗CD86(均购自TONBO Biosciences)染色以进行流式细胞术分析(BD Accuri C6，BD，美国)。
见图4。图4为实施例1得到的锰纳米佐剂(MnARK)的生物毒性、纳米佐剂被DC2.4细胞内在化以及在体外促进DC成熟实验结果。
由图4可知，与游离锰离子(氯化锰)相比，即使在浓度为0.5mmol/L时，那些MnARK纳米粒子对DC细胞也无明显的细胞毒性。同时，MnARK也可以诱导更高水平的DC细胞成熟。
以下实施例为：针对新型冠状病毒，以实施例1得到的锰纳米佐剂在纳米疫苗药物构建及免疫治疗应用。
实施例4
以实施例1得到的锰纳米佐剂结合抗原构建纳米疫苗药物，步骤如下(图5)：
A)选取新冠病毒抗原RBD作为免疫抗原研究对象。荧光标记的抗原与实施例1得到的锰纳米佐剂加入到PBS中，在25℃条件下，孵育60分钟。加入的抗原分子与锰纳米佐剂中锰元素的质量比例为1：5。
B)将步骤A得到的溶液以10,000g速度离心20分钟，收集离心下来的沉淀物即为锰纳米佐剂结合抗原所构建的纳米疫苗药物。然后通过荧光测量上 清液中未结合抗原的浓度。使用PerkinElmer荧光板读数器进行荧光测量。
参见图6。图6为实施例1所得到的锰纳米佐剂，对不同浓度抗原分子吸附数据；RBD抗原与锰纳米佐剂相互作用的缔合和解离作用实时测量的曲线；以及RBD与锰纳米佐剂(MnARK)结合后水合粒径的变化。
由图6可知，实施例1得到的纳米探针能有效吸附和载带抗原分子；RBD抗原以高亲合能稳定地与锰纳米佐剂结合；结合RBD抗原之后形成的纳米疫苗药物水合粒径对比锰纳米佐剂明显增加，说明RBD抗原与锰纳米佐剂实现了有效结合。
实施例5
A)在遵循国家动物保健协议的前提下，选取6-8周龄的BALB/c小鼠分成3组，每组12只，分别于右后腿大腿肌肉处注射以下试剂①、载带10μg荧光分子Cy5修饰的RBD抗原的25μg锰纳米佐剂构建的纳米疫苗药物；②、载带10μg荧光分子Cy5修饰的RBD抗原；③、生理盐水。
B)以注射时间点为0小时，以及12、24、48、72小时时对小鼠进行荧光成像分析。并于12、24、48、72小时荧光成像后，各取3只小鼠收集腋窝和腹股沟淋巴结进行荧光成像分析。
C)使用流式细胞仪对淋巴结组织中的DC细胞进行分析，统计RBD抗原在DC细胞中内化情况。
参见图7。图7为纳米疫苗药物活体蓄积及淋巴结靶向荧光成像。
由图7可知，与单独的RBD相比，锰纳米佐剂构建的纳米疫苗药物在注射部位诱导了更大的RBD抗原蓄积，并且抗原在该部位持续超过3天。同时，纳米疫苗药物在淋巴结中引发更强的荧光信号。定量分析显示，注射后12h，纳米疫苗药物在淋巴结中的积累效率是游离RBD抗原的约2倍，表明锰纳米佐剂构建的纳米疫苗药物促进了体内抗原向淋巴结的递送和有效蓄积。
实施例6
A)实施例4得到的纳米疫苗药物，使用R&D系统的商业试剂盒(小鼠IFN-γ酶联免疫吸附斑点(ELISPOT)装置适用试剂盒)进行ELISPOT分析。 具体过程如下，将针对小鼠IFN-γ的细胞因子捕获抗体(使用无菌PBS稀释200倍)在96孔板中包被到聚偏二氟乙烯膜(PVDF)上，并在4℃下孵育过夜。在室温下将96孔板用含有10％胎牛血清的完全1640培养基封闭2小时。分为三组，分别加入①、5μg/mL的RBD蛋白抗原；②、生理盐水；③、含有5μg/mL的RBD蛋白抗原的实施例4构建的纳米疫苗药物，随后立即将新鲜制备的小鼠脾细胞(5×10 5细胞/孔)添加到平板中。将板在37℃和5％CO 2下孵育18小时，并用补充有0.05％Tween20的PBS(PBST)洗涤四次。然后将平板与2μg/mL的针对小鼠IFN-γ的生物素化检测抗体孵育2小时。通过与抗生物素蛋白-HRP复合物在PBST中孵育一小时，然后用PBS洗涤四次，进行ELISPOT显影。最后，将板与过氧化物酶底物AEC孵育30分钟。使用自动ELISPOT读取器系统(Bio-Red)枚举ELISPOT点。
参见图8，图8为实施例4构建的纳米疫苗药物激活B细胞。
由图8可知，与仅接受RBD蛋白或生理盐水(对照组)的小鼠相比，接受实施例4构建的纳米疫苗药物的小鼠B细胞表面三种激活标志物(MHC-II，CD69和CD86)的表达显着增加，表明实施例4构建的纳米疫苗药物可以促进B细胞在体内的成熟。
实施例7
实施例4所构建的纳米疫苗药物接种。
A)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3次，每组小鼠数量为6只，一共有三组，分别是①、载带10μg RBD抗原的25μg锰纳米佐剂构建的纳米疫苗药物；②、载带50μg RBD抗原的175μg铝佐剂(Invivogen公司购买)；③、50μg RBD抗原，无纳米佐剂。以第一次小鼠大腿肌肉注射接种为第0天，第二次接种为第21天，第三次接种为第42天，并于第57天收集血清样品。
B)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，每组小鼠数量为6只，一共有三组，分别是①、载带50μg RBD抗原的25μg锰纳米佐剂构建的纳米疫苗药物，进行接种两次；②、载带50μg RBD抗原的175μg铝佐剂(Invivogen公司购买)，进行接种三次；③、50μ g RBD抗原，无纳米佐剂，进行接种三次。以第一次小鼠大腿肌肉注射接种为第0天，第二次接种为第21天，第三次接种为第42天，并于第57天收集血清样品。
C)通过传统的酶联免疫吸附测定方法(ELISA)评估步骤A及步骤B中疫苗诱导的小鼠血清中IgG和IgM水平。首先，分别用RBD抗原预涂96孔微量滴定板，在4℃孵育过夜，并在37℃用2％BSA封闭2小时。然后将步骤A及步骤B收集的小鼠血清进行梯度稀释后添加到96孔板中，然后在37℃下孵育1小时，然后用PBS洗涤四次。接着使结合的抗体在37℃下与HRP缀合的山羊抗小鼠IgG反应1小时。在用PBS洗涤四次之后，将底物3,3',5,5'-四甲基联苯胺(TMB)添加到96孔板中，并通过添加0.05％的硫酸终止反应。在ELISA板读数器(Tecan，San Jose，CA)中测量450nm和630nm处的吸光度。
见图9，图9为实施例4构建的纳米疫苗药物诱导免疫力效果对比。
由图9可知，实施例4得到的纳米疫苗药物即使在较低免疫抗原载带量(10μg)的条件下，能够比更多量的单独的RBD(50μg)或商用铝佐剂载带的RBD(50μg)，诱导产生约5倍的IgG和IgM响应强度；而抗原载带量相同的条件下，注射两次实施例4构建的纳米疫苗药物能够比注射三次单独的RBD或商用铝佐剂载带的RBD诱导产生IgG信号增强了10倍，IgM信号强度提高5倍。这些结果表明，在接受较少的抗原注射量(抗原载带量和/或注射次数)后，实施例4构建的纳米疫苗药物可产生更强的免疫反应。
实施例8
A)实施例7的步骤A得到的四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)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(5×10 4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％和90％中和抗体滴度。
B)实施例7的步骤A得到的四组小鼠血清样本进行新型冠状病毒活病毒 感染中和试验，具体来说如下：将小鼠血清以2倍梯度稀释后与活病毒混合，在37℃下孵育1小时，并一式三份加入表达ACE2的293T细胞中。每天观察每个孔的细胞病变效应(CPE)，并在感染后一周记录。计算了完全能预防CPE的小鼠抗血清中和效价。
见图10，图10为实施例4构建的纳米疫苗药物针对假病毒和新型冠状病毒活病毒的体外免疫效果对比。
由图10可知，实施例4得到的纳米疫苗药物即使在相对较低的抗体载带量的情况下，仍可诱导显著增强的针对新型冠状病毒的中和抗体应答。
以下实施例为，对比本发明所获得的锰纳米佐剂与二价锰离子(氯化锰)、透明质酸包覆的二氧化锰颗粒(Mn@HA)作为新冠疫苗佐剂的中和抗体产生效果。
实施例9
以实施例1得到的锰纳米佐剂、氯化锰盐、实验室制备透明质酸包覆二氧化锰结合抗原构建纳米疫苗药物开展动物免疫实验：
A)透明质酸包覆二氧化锰颗粒制备，称取HA为1.0g到烧瓶中，加超纯水(1g：25mL水)，到通风橱搅拌回流，温度设定为102℃，转速560rpm，注入MnCl 2溶液(1g/ml)，混匀后注入氢氧化钠(1M，15ml)，搅拌(700rpm)回流反应2h，随后超纯水透析3天，得到棕褐色的Mn@HA颗粒佐剂。
B)选取新冠病毒抗原RBD作为免疫抗原研究对象。10μg RBD抗原分别与实施例1得到的锰纳米佐剂、步骤A)到的Mn@HA颗粒、氯化锰加入到PBS中，在25℃条件下，孵育60分钟得到不同锰的佐剂疫苗。各组中加入的抗原分子与锰元素的质量比例为1：5。
C)步骤B)获得的疫苗，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3次，每组小鼠数量为6只，一共有五组，分别是①、载带10μg RBD抗原的25μg锰纳米佐剂构建的纳米疫苗药物；②、载带10μg RBD抗原的25μg Mn@HA佐剂构建的疫苗；③、10μg RBD抗原的25μg氯化锰构建的疫苗；④、10μg RBD抗原，无纳米佐剂；⑤、生理盐水组。以第一次小鼠大腿肌肉注射接种为第0天，第二次接种为第21天， 第三次接种为第42天，并于第57天收集血清样品。通过传统的酶联免疫吸附测定方法(ELISA)评估步骤A及步骤B中疫苗诱导的小鼠血清中IgG水平。
由图11可知，与游离RBD相比，所有三种基于Mn的疫苗佐剂均显示RBD特异性IgG水平显着增加。值得注意的是，在锰纳米疫苗组中发现了最高的IgG水平，这表明锰纳米疫苗可以在体内引发强大的免疫反应，并且优于MnCl 2和Mn@HA。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 四氧化三锰颗粒在制备锰纳米佐剂中应用，其特征在于，所述锰纳米佐剂包括四氧化三锰纳米颗粒和包裹于其外的模板分子，模板分子与锰元素的摩尔比为1：(10～1000)，所述模板分子包括模板蛋白及其片段或多肽。
  2. 根据权利要求1所述的应用，其特征在于，所述模板蛋白包括牛血清白蛋白、人血清白蛋白、鼠血清白蛋白、转铁蛋白、抗原蛋白中的一种或几种；所述多肽包括上述模板蛋白衍生片段、抗原多肽。
  3. 根据权利要求1或2所述的应用，其特征在于，所述锰纳米佐剂的平均粒径为1～100nm。
  4. 一种锰纳米佐剂的制备方法，其特征在于，包括如下步骤：
    (1)将二价锰盐水溶液与模板分子溶液进行混合，得到复合溶液；
    (2)调节复合溶液pH值至9或9以上，搅拌，透析纯化，冻干，得到锰纳米佐剂；
    所述模板分子包括模板蛋白及其片段或多肽。
  5. 根据权利要求4所述的制备方法，其特征在于，所述二价锰盐选自氯化锰、硝酸锰、乙酸锰、硫酸锰中的一种或几种；调节复合溶液pH值的试剂为碱性试剂，碱性试剂为氢氧化钠、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；所述复合溶液中，二价锰盐的浓度为0.1～0.5mol/L，模板蛋白的浓度为1～50g/L。
  6. 根据权利要求4至5中任一项所述的制备方法，其特征在于，所述搅拌的温度为30～37℃，搅拌的时间为1～10小时，搅拌的转速为200～1000r/min。
  7. 由权利要求4至6任一所述方法制备的锰纳米佐剂。
  8. 锰纳米佐剂在制备疫苗中的应用，其特征在于，所述疫苗包括权利要求1至7中任一所述所述的锰纳米佐剂。
  9. 一种疫苗，其特征在于，包括抗原和权利要求1至7中任一所述的锰纳米佐剂，所述抗原与锰纳米佐剂中锰元素的质量比例为1：(0.025～40)。
  10. 权利要求9所述疫苗的制备方法，其特征在于，将锰纳米佐剂与抗原在20～37℃的缓冲液中孵育30～120分钟。
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