WO2022226671A1 - 一种特异性预防真菌感染的寡糖疫苗及其制备方法 - Google Patents

一种特异性预防真菌感染的寡糖疫苗及其制备方法 Download PDF

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WO2022226671A1
WO2022226671A1 PCT/CN2021/000153 CN2021000153W WO2022226671A1 WO 2022226671 A1 WO2022226671 A1 WO 2022226671A1 CN 2021000153 W CN2021000153 W CN 2021000153W WO 2022226671 A1 WO2022226671 A1 WO 2022226671A1
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oligosaccharide
vaccine
protein
fungal infection
preparation
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French (fr)
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刘飞
袁丹丹
陈磊
牛林林
陈勉
邵华荣
张小刚
刘英梅
张金华
刁梦奇
郭新艳
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山东省药学科学院
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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
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61P31/10—Antimycotics
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an oligosaccharide vaccine for specifically preventing fungal infection and a preparation method thereof.
  • Fungi are ubiquitous microorganisms in nature and common bacteria that colonize the surface of human skin and mucous membranes.
  • fungi can invade the human body and cause lethal infections.
  • lethal infections According to incomplete statistics, more than 400 of the 1.5 million species of fungi in the world can cause serious human infections.
  • Most systemic fungal diseases are caused by Candida, Cryptococcus, and Aspergillus. Compared with other microbial infections, the clinical understanding of systemic fungal infections is not deep, the diagnostic methods are limited, and many patients cannot receive timely treatment.
  • the thiolated chitosan oligosaccharide was linked with a heterobifunctional linker to form an adjuvant, and then coupled with attenuated vaccines, protein vaccines, etc.
  • the chitosan oligosaccharide adjuvant can enhance the immune response, but because the chitosan oligosaccharide contains
  • a multi-site thiolated chitosan oligosaccharide is conjugated with multiple vaccines, and the multiple amino epitopes equivalent to chitosan oligosaccharide are modified, which is no longer the complete component of the fungal cell wall. institutions, and the vaccine prepared by it cannot elicit an immune response that effectively recognizes the fungus.
  • Chitin is a component that widely exists in the fungal cell wall.
  • Chitin oligosaccharide is a small molecule oligosaccharide obtained by enzymatic hydrolysis of chitin after deacetylation.
  • Chitin oligosaccharide is a small molecule oligosaccharide obtained by enzymatic hydrolysis of chitin.
  • all have a monosaccharide composition similar to chitin, and can be used as a broad-spectrum antifungal target with certain immunogenicity.
  • no broad-spectrum fungal vaccine has been reported in this field, and the mechanism of its recognition by the immune system remains unclear. Therefore, the research of antifungal vaccine based on fungal cell structure has very urgent practical significance.
  • the object of the present invention is to provide a vaccine for the specific prevention of fungal infection, which is used for the prevention and treatment of infection caused by fungi in immunosuppressed patients.
  • a first aspect of the present invention provides a specific oligosaccharide vaccine for preventing fungal infection and a preparation method thereof, comprising:
  • the carrier protein containing primary amino group is thiolated to obtain thiolated protein
  • the thiolated protein is coupled with the chitosan oligosaccharide mixture and/or the chitin oligosaccharide mixture to form an oligosaccharide vaccine.
  • the oligosaccharide vaccine formed by coupling the sulfhydryl-modified protein with the oligosaccharide has a clear coupling position and strong specificity; at the same time, the oligosaccharide used is a mixture of chitosan oligosaccharides and/or chitin with different degrees of polymerization
  • the oligosaccharide mixture is more likely to induce immune response of body cells; therefore, the final oligosaccharide vaccine can better identify and prevent infections caused by fungi (such as Candida albicans, Aspergillus, Cryptococcus neoformans, etc.).
  • the second aspect of the present invention provides an oligosaccharide vaccine for specific prevention of fungal infection prepared by any of the above methods.
  • the third aspect of the present invention provides the use of the above-mentioned specific oligosaccharide vaccine for preventing fungal infection in preparing a drug for treating or preventing systemic fungal infection, or a drug for activating Th17 cell immune response.
  • the oligosaccharide vaccine provided by the present invention is to introduce a sulfhydryl group (-SH) into a carrier protein containing a primary amino group (-NH 2 ) to form a sulfhydrylated protein, which is then coupled with the oligosaccharide through the linking action of a bridging agent Form oligosaccharide vaccine.
  • the coupling position of the oligosaccharide vaccine is clear, and it is coupled to the reducing end group (free aldehyde group, -CHO) of the oligosaccharide, which maintains the original structural characteristics of the oligosaccharide and has strong specificity.
  • Chitosan oligosaccharide is a mixture of different degrees of polymerization, that is, by increasing the diversity including the degree of polymerization and the distribution of acetyl groups, the prepared vaccine is highly immunogenic, can activate Th17 cell immune responses, and can recognize and prevent fungi (such as Candida albicans). bacteria, Aspergillus, Cryptococcus neoformans, etc.).
  • SATA refers to: N-succinimidyl-S-acetylthioacetate
  • PDPH refers to: 3-(2-pyridyldithio)propanohydrazide.
  • oligosaccharide vaccine for specific prevention of fungal infection
  • the vaccine is a conjugate of oligosaccharide and thiolated protein.
  • Thiolated protein is the introduction of sulfhydryl group (-SH) into the carrier protein containing primary amino group (-NH 2 ), and then through the linking effect of bridging agent, it is coupled with oligosaccharide to form oligosaccharide vaccine, and the coupling of oligosaccharide and carrier protein.
  • the combined molar ratio is (200-500): 1 or more.
  • the oligosaccharide is a chitosan oligosaccharide mixture and/or a chitin oligosaccharide mixture, both of which are mixtures of different degrees of polymerization, the relative molecular mass is less than or equal to 5000 Da, and the degree of deacetylation is 0-100%.
  • Described protein is non-human protein, selected from Concholepasconcholepas hemocyanin (CCH), keyhole limpet hemocyanin (KLH, keyhole limpet hemocyanin), bovine serum albumin (BSA), tetanus toxin/toxoid, rotavirus VP7
  • CCH Concholepasconcholepas hemocyanin
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxin/toxoid rotavirus VP7
  • CCH Concholepasconcholepas hemocyanin
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxin/toxoid rotavirus VP7
  • diphtheria toxin mutant CRM Bacillus perfringens exotoxin/toxoid.
  • the preparation method of described thiolated protein comprises the following steps:
  • step (4) of the preparation method of thiolated protein the addition amount of the hydroxylamine solution is 100 ⁇ l of hydroxylamine solution per milliliter of protein solution.
  • the coupling of the oligosaccharide and the thiolated protein comprises the following steps:
  • the bridging agent is PDPH.
  • the molar concentration of the oligosaccharide is equivalent to 100-1200 times the molar amount of the thiolated protein.
  • the method for removing impurity small molecules is dialysis or column chromatography.
  • the buffer used for column chromatography or dialysis is the solution used to dissolve the carrier protein.
  • the packing type of the desalting column is one of Sephadex G10-G50, Bio-gel P2-P10, Thermo Scientific TM Zeba TM Spin Desalting Columns.
  • the molecular weight cut-off of the dialysis bag of the dialysis method is 1000-10000 Da, and the flattening width is 1-3.5 cm.
  • the phosphate buffer mentioned above is 0.01-0.1M sodium phosphate, 0-0.15M sodium chloride, pH 7.0-8.0.
  • oligosaccharide vaccine is used for the treatment or prevention of infection caused by systemic fungi, including but not limited to Candida albicans, Aspergillus, and Cryptococcus neoformans.
  • SATA SATA to DMF to prepare a 2 mg/ml SATA/DMF solution.
  • 2 ml of 2 mg/ml carrier protein (BSA) solution was prepared with 0.01 M sodium phosphate (pH 7.0) buffer, 5 times more molar amount of SATA/DMF solution was added, and the reaction was carried out at room temperature for 30 minutes.
  • Hydroxylamine ⁇ HCl was added to 0.01 M sodium phosphate (pH 7.0) buffer to prepare a 20 mg/ml hydroxylamine solution.
  • PDPH was added to DMF to prepare a 5mM PDPH/DMF solution.
  • the small impurity molecules were removed through a dialysis bag with a molecular weight cut-off of 5000 Da to obtain a chitosan oligosaccharide-carrier protein conjugate, which was vacuum freeze-dried to obtain an oligosaccharide vaccine.
  • SATA SATA to DMF to prepare a 3 mg/ml SATA/DMF solution.
  • CHC carrier protein
  • pH7.2 0.01M sodium phosphate and 0.01M sodium chloride
  • Hydroxylamine ⁇ HCl was added to 0.01 M sodium phosphate, 0.01 M sodium chloride (pH 7.2) buffer to prepare a 40 mg/ml hydroxylamine solution. A certain amount of hydroxylamine solution was added to the SATA-protein solution and reacted at room temperature for 1 hour to obtain a thiolated protein solution.
  • PDPH was added to DMF to prepare a 20 mM PDPH/DMF solution.
  • SATA SATA to DMF to prepare a 5 mg/ml SATA/DMF solution.
  • Hydroxylamine ⁇ HCl was added to a 0.1 M sodium phosphate, 0.01 M sodium chloride (pH 7.2) buffer to prepare a 100 mg/ml hydroxylamine solution.
  • PDPH was added to DMF to prepare a 30 mM PDPH/DMF solution. Add PDPH in excess of 30 times the molar amount to the thiolated protein solution prepared in the above (1), and react at room temperature for 3 hours. Chitosan oligosaccharide with a degree of deacetylation of 90% and a molecular weight of ⁇ 2000Da, which is equivalent to 800 times the molar amount of protein, was added, and the reaction was carried out at 4°C for 3 days.
  • SATA SATA to DMF to prepare a 6 mg/ml SATA/DMF solution.
  • Hydroxylamine ⁇ HCl was added to a 0.1 M sodium phosphate, 0.01 M sodium chloride (pH 7.2) buffer to prepare a 100 mg/ml hydroxylamine solution.
  • PDPH was added to DMF to prepare a 50 mM PDPH/DMF solution.
  • SATA SATA to DMF to prepare a 5 mg/ml SATA/DMF solution.
  • 10mg/ml carrier protein (diphtheria toxin mutant CRM) solution with 0.1M sodium phosphate and 0.15M sodium chloride buffer at pH 8.0, add SATA/DMF solution in excess of 15 times the molar amount, and react at room temperature 30 minutes.
  • Hydroxylamine ⁇ HCl was added to 0.1M sodium phosphate and 0.15M sodium chloride buffer at pH 8.0 to prepare a 60 mg/ml hydroxylamine solution.
  • PDPH was added to DMF to prepare a 40 mM PDPH/DMF solution.
  • PDPH was added to DMF to prepare an 80 mM PDPH/DMF solution.
  • the small impurity molecules were removed through a dialysis bag with a molecular weight cut-off of 10000Da to obtain a chitosan oligosaccharide-carrier protein conjugate, which was vacuum frozen Dried oligosaccharide vaccine.
  • Example 6 the coupling ratio of the chromatographic fluid sample was detected, and the coupling ratio was the number of moles of chitosan oligosaccharide attached to each mole of carrier protein.
  • glucosamine hydrochloride dissolve it in a 10ml volumetric flask with purified water, adjust the volume to the mark and shake well to obtain a standard solution mother liquor of 100mg/ml, store in a refrigerator at 4°C away from light, Valid for two weeks. Pipette 0, 5, 10, 20, 30, 40 ⁇ L of glucosamine standard solution into a 1.5 ml centrifuge tube, and make up to 1 ml with purified water to obtain standard solutions of different concentrations. At the same time, 5, 10 and 20 mg/ml chitosan oligosaccharide solutions were prepared with purified water.
  • glucosamine hydrochloride in (1) Take 100 mg/ml glucosamine hydrochloride in (1) as the standard solution mother solution, draw 0, 10, 30, 60, 90, 120 ⁇ L of glucosamine standard solution into a 1.5 ml centrifuge tube, and make up to 1 ml with purified water to obtain standard solutions of different concentrations. At the same time, 5, 10 and 20 mg/ml chitosan oligosaccharide solutions were prepared with purified water.
  • Degree of polymerization of chitosan oligosaccharide moles of total chitosan oligosaccharides/moles of chitosan oligosaccharide reducing sugars
  • Coupling ratio moles of chitosan oligosaccharides attached to protein/(10 ⁇ moles of protein)
  • Immunocompromised (neutropenic) mouse model was used to study the effectiveness of the vaccine, this neutropenic mouse model is considered to be a suitable model to simulate the effects of chemotherapy for various malignancies, etc. patients with this defect.
  • Immunity-compromised mice injected with cyclophosphamide increased susceptibility to the fungus, similar to the increased susceptibility to the disease in neutropenic patients.
  • the immunogenicity of the chitooligosaccharide-carrier protein vaccine was evaluated by taking the blood of mice to detect the changes of the cytokine IL-17F in Th17 cells.
  • mice 6-8 week old Balb/c female mice were selected and fed adaptively for 3-7 days.
  • 20g/L cyclophosphamide was prepared with normal saline, and cyclophosphamide was injected intraperitoneally at 200mg/kg body weight.
  • the number of neutrophils and neutrophils in the blood of mice were measured by SYSMEX XT-2000i automatic blood analyzer. ⁇ 500/mm 3 , the experiment can be continued, and then cyclophosphamide is injected every 10 days at a dose of 150 mg/kg and maintained for the entire experimental period.
  • mice were injected subcutaneously at multiple points on the neck and back at a dose of 2.5 mg/kg.
  • the normal saline blank group was injected with normal saline, and the fluconazole group was not injected.
  • mice were given intragastrically at 50 mg/kg ⁇ d for 7 consecutive days, and the test samples containing adjuvant were injected in other groups.
  • Freund's adjuvant In order to stimulate the body to produce a strong immune response, Freund's adjuvant is used. Freund's complete adjuvant was used for immunization injection in week 0, and incomplete Freund's adjuvant was used for immunization injection in week 2, 4, and 6. KLH, chitooligosaccharide and chitooligosaccharide-KLH were prepared into 1 mg/mL solution with 0.02M PBS (pH7.2-7.4) buffer, and then mixed with equal volume of Freund's complete adjuvant or incomplete Freund's adjuvant, Homogenize by ultrasonic emulsification.
  • Extracellular staining Centrifuge the 96-well plate at 1600 rpm for 10 min (same as the centrifugation conditions), discard the supernatant, and add 70 ⁇ L/well of FACS buffer containing extracellular staining antibodies (containing fluorescent staining CD3e, CCR6, CD4, 7-AAD antibody, added in an amount of 1:100, 1:100, 1:200, 1:100), pipetting, and incubated in the dark for 40 min for extracellular staining.
  • extracellular staining antibodies containing fluorescent staining CD3e, CCR6, CD4, 7-AAD antibody, added in an amount of 1:100, 1:100, 1:200, 1:100
  • Membrane rupture Add 180 ⁇ L/well of FACS buffer without antibody and pipette, centrifuge to discard the supernatant, add 100 ⁇ L/well of membrane rupture solution, and incubate in the dark at 4°C for 40 min to perform membrane rupture treatment.
  • Intracellular staining pipetting with 1 ⁇ 180 ⁇ L/well of membrane-breaking buffer, centrifuging to discard the supernatant, adding 70 ⁇ L/well of membrane-breaking buffer containing intracellular staining antibody, and incubating at room temperature for 40 minutes in the dark for intracellular staining. Cytokine staining.
  • Cytokine detection add FACS buffer at 180 ⁇ L/well and pipet it, centrifuge to discard the supernatant, add FACS buffer at 100 ⁇ L/well and pipet it, and measure the level of IL-17F in Th17 cells by flow cytometry, which reflects Activation of test samples on cellular immunity in mice.
  • the ratio of intracellular IL-17F to Th17 before and after immunization of mice in each group is shown in Table 3.
  • the proportion of intracellular IL-17F in mice in the oligosaccharide-KLH group was significantly increased (P ⁇ 0.01), indicating that the oligosaccharide coupled with carrier protein can stimulate Th17 cell immunity, promote the secretion of cytokine IL-17F, to antifungal effect.
  • Candida albicans as the challenge strain, the protective effect of oligosaccharide vaccine on fungal challenge was studied.
  • the cultured Candida albicans was suspended in 0.01M PBS, and the concentration was adjusted to 0.5 ⁇ 10 6 , 1.0 ⁇ 10 6 , 0.5 ⁇ 10 7 , 1.0 ⁇ 10 7 , 0.5 ⁇ 10 8 , 1.0 ⁇ 10 8 CFU/ml , at a dose of 0.1 ml/mouse, the mice were challenged by tail vein injection, and the mice were selected from immunocompromised mice (the number of neutrophils ⁇ 500/mm 3 ) injected with cyclophosphamide in step 1 of Example 8, and set at the same time. In the normal saline control group, the mortality of mice within 5 days was counted to determine the minimum lethal dose. The results are shown in Table 4.
  • mice in the control group did not die.
  • the mice in the 0.5 ⁇ 10 5 CFU and 1.0 ⁇ 10 5 CFU dose groups did not die within 5 days; the 0.5 ⁇ 10 6 CFU dose group had a mortality rate of 30% within 5 days; the 0.5 ⁇ 10 7 CFU dose group had a 5-day mortality rate of 100%; The mortality rate within 12 hours of the 1.0 ⁇ 10 7 CFU dose group was 100%. It can be seen that the minimum lethal dose of Candida albicans to mice was 0.5 ⁇ 10 7 CFU.
  • the survival rate of mice was calculated after 7 days, as shown in Table 5.

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Abstract

一种特异性预防真菌感染的寡糖疫苗及其制备方法，该疫苗是由巯基化蛋白与寡糖偶联而成，巯基化蛋白是将巯基(-SH)引入含有伯氨基(-NH2)的载体蛋白上，然后通过桥联剂的联结作用，与寡糖偶联，形成寡糖疫苗。所述的载体蛋白为非人源蛋白，寡糖为壳寡糖混合物和/或几丁质寡糖混合物。所述疫苗免疫原性强，能够激活Th17细胞免疫，可以识别并保护真菌所致的感染。

Description

一种特异性预防真菌感染的寡糖疫苗及其制备方法 技术领域
本发明属于生物技术领域，具体涉及一种特异性预防真菌感染的寡糖疫苗及其制备方法。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
真菌是自然界中广泛存在的微生物，也是人类皮肤、黏膜表面定居的常见菌，然而，当免疫系统受损或宿主屏障遭到破坏时，真菌可以侵入人体，导致致死性感染。据不完全统计，全球150万种真菌中有400多种能够导致人类严重的感染，大多数系统性真菌病是由念珠菌(Candida)、隐球菌(Cryptococcus)、曲霉菌(Aspergillus)引起的，相比于其它微生物的感染，临床上对于系统性真菌感染的认识不深、诊断方法有限，许多病人得不到及时的治疗。
近年来，尽管抗真菌药物越来越多，但因为抗真菌药物的抗菌谱有限、毒副作用和真菌的耐药性等问题，抗真菌药物的疗效是有限的。研究表明，与传统的抗真菌药物相比，真菌疫苗的开发显示了良好的应用前景。以β-葡聚糖、合成甘露糖与载体蛋白偶联后的糖蛋白化合物作为疫苗，起到了预防侵袭性真菌感染效果，但由于受试抗原需要通过复杂的化学合成工艺，限制了可用于筛选有效糖抗原的品种数量，目前在研疫苗的保护作用仍有待提高。另有研究将巯基化壳寡糖与异型双功能连接剂连接形成佐剂，再与减毒疫苗、蛋白质疫苗等偶联，此壳寡糖佐剂能够增强免疫应答，但由于壳寡糖上含有多个氨基，被巯基化后，一个多位点巯基化的壳寡糖与多个疫苗偶联，相当于壳寡糖的多个氨基抗原决定簇遭到修饰，不再是真菌细胞壁成分的完整机构，其制备的疫苗不能激发出有效识别真菌的免疫应答。
几丁质是广泛存在于真菌细胞壁的成分，壳寡糖是几丁质脱乙酰基后酶解得小分子寡糖，几丁质寡糖是几丁质经过酶解后得到的小分子寡糖，均具有与几丁质相似的单糖组成，可作为广谱抗真菌的靶点，具有一定的免疫原性。而该领域的广谱真菌疫苗还没有报道，其被免疫系统识别等机制仍不清楚。因此，基于真菌细胞结构的抗真菌疫苗的研究具有非常迫切的现实意义。
发明内容
为了克服上述问题，本发明的目的是提供一种特异性预防真菌感染的疫苗，用于预防和治疗免疫抑制患者真菌引起的感染。
为实现上述技术目的，本发明采用如下技术方案：
本发明的第一个方面，提供了一种特异性预防真菌感染的寡糖疫苗及其制备方法，包括：
对含有伯氨基的载体蛋白进行巯基化修饰，得到巯基化蛋白；
将所述巯基化蛋白与壳寡糖混合物和/或几丁质寡糖混合物偶联，形成寡糖疫苗。
研究发现：将巯基化修饰的蛋白与寡糖偶联，形成的寡糖疫苗偶联位置明确，特异性强；同时，采用的寡糖是不同聚合度的壳寡糖混合物和/或几丁质寡糖混合物，更容易引起机体细胞的免疫应答；因此，最终获得的寡糖疫苗可以更好地识别并预防真菌(如白色念珠菌、曲霉菌、新型隐球菌等)所致的感染。
本发明的第二个方面，提供了任一上述的方法制备的特异性预防真菌感染的寡糖疫苗。
本发明的第三个方面，提供了上述的特异性预防真菌感染的寡糖疫苗在制备治疗或预防系统性真菌感染的药物、或激活Th17细胞免疫应答的药物中的应用。
本发明的有益效果在于：
(1)本发明提供的寡糖疫苗是将巯基(-SH)引入含有伯氨基(-NH 2)的载体蛋白上，形成巯基化蛋白，然后通过桥联剂的联结作用，与寡糖偶联形成寡糖疫苗。寡糖疫苗的偶联位置明确，偶联于寡糖的还原端基(游离醛基，-CHO)，保持了寡糖原有结构特征，特异性强，其中，使用的壳寡糖和/或几丁寡糖为不同聚合度的混合物，即通过增加包括聚合度、乙酰基分布的多样性，使制备的疫苗免疫原性强，能够激活Th17细胞免疫应答，可以识别并预防真菌(如白色念珠菌、曲霉菌、新型隐球菌等)所致的感染。
(2)本申请的操作方法简单、成本低、具有普适性，易于规模化生产。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
术语解释
本申请中，SATA是指：N-琥珀酰亚胺基-S-乙酰硫基乙酸酯；
本申请中，PDPH是指：3-(2-吡啶基二硫代)丙酰肼。
一种特异性预防真菌感染的寡糖疫苗，所述疫苗为寡糖与巯基化蛋白的偶联体。巯基化 蛋白是将巯基(-SH)引入含有伯氨基(-NH 2)的载体蛋白上，然后通过桥联剂的联结作用，与寡糖偶联形成寡糖疫苗，寡糖与载体蛋白的偶联摩尔比达(200-500)∶1以上。
所述的寡糖为壳寡糖混合物和/或几丁质寡糖混合物，均为不同聚合度的混合物，相对分子质量≤5000Da，脱乙酰度为0-100％。
所述的蛋白为非人源蛋白，选自Concholepasconcholepas hemocyanin(CCH)、钥孔血蓝蛋白(KLH，keyhole limpet hemocyanin)、牛血清白蛋白(BSA)、破伤风毒素/类毒素、轮状病毒VP7蛋白、白喉毒素突变体CRM、产气荚膜羧状芽胞杆菌外毒素/类毒素中的一种。
所述的巯基化蛋白的制备方法包括如下步骤：
(1)将SATA中加入二甲基甲酰胺(DMF)中，制备2-10mg/ml SATA/DMF溶液。
(2)用磷酸盐缓冲液配制1-10mg/ml蛋白溶液，加入超过其5-100倍摩尔量的SATA/DMF溶液，在室温下反应20-80分钟。
(3)将羟胺·HCl加入磷酸盐缓冲液中，制备10-100mg/ml羟胺溶液。
(4)在SATA-蛋白溶液中加入一定量的羟胺溶液，在室温下反应0.5-4小时，得到巯基化蛋白溶液。
巯基化蛋白的制备方法步骤(4)中，所述羟胺溶液的添加量为每毫升蛋白溶液加入100μl的羟胺溶液。
所述的寡糖与巯基化蛋白的偶联包括以下步骤：
(1)将桥联剂加入二甲基甲酰胺(DMF)中，制备5-100mM桥联剂溶液。
(2)在巯基化蛋白的制备方法中的步骤(4)制备得到的巯基化蛋白溶液中加入超过其5-50倍摩尔量的桥联剂，室温反应1-6小时。
(3)加入寡糖，4℃反应1-7天。
(4)去除杂质小分子，得寡糖疫苗。
巯基化蛋白与寡糖的偶联步骤(1)(2)中，所述的桥联剂为PDPH。
寡糖与巯基化蛋白的偶联步骤(3)中，所述的寡糖的摩尔浓度相当于巯基化蛋白摩尔量的100-1200倍。
寡糖与巯基化蛋白的偶联步骤(4)中，所述去除杂质小分子的方法为透析或柱层析。柱层析或透析所用缓冲液为溶解载体蛋白时所用的溶液。所述脱盐柱的填料型号为Sephadex G10-G50、Bio-gel P2-P10、Thermo Scientific TMZeba TM Spin Desalting Columns中的一种。所述透析法的透析袋的截留分子量为1000-10000Da，压平宽度为1-3.5cm。
以上所述磷酸盐缓冲液为0.01-0.1M的磷酸钠，0-0.15M的氯化钠，pH7.0-8.0。
上述寡糖疫苗作为治疗或预防系统性真菌所致的感染，所述真菌包括但不限于白色念珠菌、曲霉菌、新型隐球菌。
下面结合具体的实施例，对本发明做进一步的详细说明，应该指出，所述具体实施例是对本发明的解释而不是限定。
实施例1
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备2mg/ml SATA/DMF溶液。用0.01M的磷酸钠(pH7.0)缓冲液配制2mg/ml载体蛋白(BSA)溶液2ml，加入超过其5倍摩尔量的SATA/DMF溶液，在室温下反应30分钟。将羟胺·HCl加入0.01M的磷酸钠(pH7.0)缓冲液中，制备20mg/ml羟胺溶液。在SATA-蛋白溶液中加入200μL羟胺溶液，在室温下反应2小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备5mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其5倍摩尔量的PDPH，室温反应1h。加入相当于蛋白摩尔量100倍的脱乙酰度为85％、分子量≤5000Da壳寡糖，4℃反应1天。以0.01M氯化钠(pH7.0)为透析用缓冲液，通过截流分子量5000Da透析袋除去杂质小分子，得到壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例2
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备3mg/ml SATA/DMF溶液。用0.01M磷酸钠、0.01M氯化钠(pH7.2)缓冲液配制4mg/ml载体蛋白(CCH)溶液3ml，加入超过其80倍摩尔量的SATA/DMF溶液，在室温下反应50分钟。以0.01M磷酸钠、0.01M氯化钠(pH7.2)为透析用缓冲液，通过截流分子量10000Da透析袋除去杂质小分子，收集透析液，得到SATA-蛋白溶液。将羟胺·HCl加入0.01M磷酸钠、0.01M氯化钠(pH7.2)缓冲液中，制备40mg/ml羟胺溶液。在SATA-蛋白溶液中加入一定量羟胺溶液，在室温下反应1小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备20mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其15倍摩尔量的PDPH，室温反应1h，透析，收集透析液。加入相当于蛋白摩尔量500倍的脱乙酰度为5％、分子量≤2000Da甲壳寡糖，4℃反应2天。透析除 去杂质小分子，得到甲壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例3
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备5mg/ml SATA/DMF溶液。用0.1M磷酸钠、0.01M氯化钠(pH7.2)缓冲液配制6mg/ml载体蛋白(KLH)溶液2ml，加入超过其100倍摩尔量的SATA/DMF溶液，在室温下反应50分钟。将羟胺·HCl加入以0.1M磷酸钠、0.01M氯化钠(pH7.2)缓冲液中，制备100mg/ml羟胺溶液。在SATA-蛋白溶液中加入200μL羟胺溶液，在室温下反应3小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备30mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其30倍摩尔量的PDPH，室温反应3h。加入相当于蛋白摩尔量800倍的脱乙酰度为90％、分子量≤2000Da壳寡糖，4℃反应3天。以0.1M磷酸钠、0.01M氯化钠(pH7.2)的缓冲液为流动相，通过Bio-gel P2 Desalting Columns，样品通过柱子后，开始接样品，10滴(约0.5ml)一管，连续接20管，经检测后得到壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例4
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备6mg/ml SATA/DMF溶液。用0.1M磷酸钠、0.01M氯化钠(pH7.2)缓冲液配制8mg/ml载体蛋白(破伤风毒素)溶液2ml，加入超过其30倍摩尔量的SATA/DMF溶液，在室温下反应30分钟。将羟胺·HCl加入以0.1M磷酸钠、0.01M氯化钠(pH7.2)缓冲液中，制备100mg/ml羟胺溶液。在SATA-蛋白溶液中加入200μL羟胺溶液，在室温下反应3小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备50mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其30倍摩尔量的PDPH，室温反应3h。加入相当于蛋白摩尔量200倍的脱乙酰度为2％、分子量≤1000Da甲壳寡糖，4℃反应4天。以0.1M磷酸钠、0.01M氯化钠(pH7.2)的缓冲液为流动相，通过拖盐Bio-gel P2，样品通过柱子后，开始接样品，10滴(约0.5ml)一管，连续接20管，经检测后得到甲壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例5
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备5mg/ml SATA/DMF溶液。用pH8.0的0.1M磷酸钠、0.15M氯化钠缓冲液配制10mg/ml载体蛋白(白喉毒素突变体CRM)溶液2ml，加入超过其15倍摩尔量的SATA/DMF溶液，在室温下反应30分钟。将羟胺·HCl加入pH8.0的0.1M磷酸钠、0.15M氯化钠缓冲液中，制备60mg/ml羟胺溶液。在SATA-蛋白溶液中加入200μL羟胺溶液，在室温下反应2小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备40mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其20倍摩尔量的PDPH，室温反应4h。加入相当于蛋白摩尔量200倍的脱乙酰度为95％、分子量≤1000-3000Da壳寡糖，4℃反应3天。以pH8.0的0.1M磷酸钠、0.15M氯化钠缓冲液为流动相，通过脱盐柱Thermo Scientific TMZeba TM Spin，样品通过柱子后，开始接样品，10滴(约0.5ml)一管，连续接20管，经检测后得到壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例6
(1)巯基化蛋白的制备
将SATA中加入DMF中，制备10mg/ml SATA/DMF溶液。用pH7.2的0.1M磷酸钠、0.15M氯化钠缓冲液配制6mg/ml载体蛋白(KLH)溶液2ml，加入超过其70倍摩尔量的SATA/DMF溶液，在室温下反应30分钟。将羟胺·HCl加入pH7.2的0.1M磷酸钠、0.15M氯化钠缓冲液中，制备100mg/ml羟胺溶液。在SATA-蛋白溶液中加入200μL羟胺溶液，在室温下反应3小时，得到巯基化蛋白溶液。
(2)寡糖与巯基化蛋白的偶联
将PDPH加入DMF中，制备80mM PDPH/DMF溶液。将上述(1)中制备得到的巯基化蛋白溶液中加入超过其50倍摩尔量的PDPH，室温反应5h。加入相当于蛋白摩尔量900倍的脱乙酰度为98％、分子量≤3000Da壳寡糖，4℃反应4天。以pH7.2的0.1M磷酸钠、0.15M氯化钠缓冲液为流动相为透析用缓冲液，通过截流分子量10000Da透析袋除去杂质小分子，得到壳寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例7寡糖-巯基化蛋白偶联程度测定
以实施例6为例，检测层析液样品偶联比，偶联比为每摩尔载体蛋白上连接的壳寡糖摩尔数。
(1)样品中还原糖(游离壳寡糖)含量测定
称取氨基葡萄糖盐酸盐1g(精确至0.001g)，用纯化水溶解于10ml容量瓶中，定容至刻度后摇匀，得100mg/ml的标准溶液母液，4℃冰箱中避光贮存，两周内有效。分别吸取0、5、10、20、30、40μL氨基葡萄糖标准溶液于1.5ml离心管中，用纯化水补至1ml，得不同浓度标准溶液。同时用纯化水配制5、10、20mg/ml壳寡糖溶液。
吸取标准品或待测溶液30μL，加入30μLDNS，混匀，沸水浴5min，自来水冷却，加180μL纯化水，混匀，取200μL加入96孔板，酶标仪测定吸光度OD 540。以氨基葡萄糖盐酸盐摩尔浓度为横坐标，吸光度为纵坐标，制定标准曲线，得标准曲线y＝58.031x-0.0167，R 2＝0.9981。以标准曲线测定样品及壳寡糖溶液中游离壳寡糖的浓度。
(2)样品中总糖含量测定
以(1)中100mg/ml氨基葡萄糖盐酸盐为标准溶液母液，分别吸取0、10、30、60、90、120μL氨基葡萄糖标准溶液于1.5ml离心管中，用纯化水补至1ml，得不同浓度标准溶液。同时用纯化水配制5、10、20mg/ml壳寡糖溶液。
取0.1ml样品或标准品，加入0.3ml蒽酮-硫酸溶液，沸水浴10min，立即放入冰水中15min，取200μL放入96孔板中，酶标仪测定吸光度OD 620。以氨基葡萄糖盐酸盐摩尔浓度为横坐标，吸光度为纵坐标，制定标准曲线，得标准曲线y＝20.198x-0.017，R 2＝0.9977。以标准曲线测定样品及壳寡糖溶液中总糖的浓度。
(3)Bradford法测蛋白含量
配制5mg/ml BSA标准溶液母液，配成0、0.02、0.05、0.1、0.3、0.5、0.7mg/ml的标准溶液。
取标准品或10倍稀释样品20μL加入96孔板中，各孔加入200μL考马斯亮蓝1×G250染色液，室温放置3-5min，用酶标仪测定吸光度OD 595。以BSA浓度为横坐标，吸光度为纵坐标，制定标准曲线，得标准曲线y＝2.1838x+0.0294，R 2＝0.9948。以标准曲线测定样品中蛋白的浓度。
(4)偶联比的计算
壳寡糖的聚合度＝壳寡糖总糖摩尔数/壳寡糖还原糖摩尔数
连在蛋白上的壳寡糖的摩尔数mmol/ml＝(总糖-还原糖×聚合度)/聚合度
偶联比＝连在蛋白上的壳寡糖的摩尔数/(10×蛋白的摩尔数)
(5)结果分析
通过还原糖测定发现，样品透析后无颜色反应，说明无还原糖(游离壳寡糖)存在；总糖测定显示，透析后的样品显棕色，吸光值较大，说明有糖存在；Bradford法测蛋白，透 析后样品具有明显的颜色反应，显亮蓝色，吸光值较大。以上结果表明壳寡糖与蛋白发生了偶联。根据测定数据，壳寡糖的聚合度如表1所示，计算得出样品偶联比为2395，数据如表2所示。
表1壳寡糖的聚合度
Figure PCTCN2021000153-appb-000001
表2壳寡糖与载体蛋白的偶联数据
Figure PCTCN2021000153-appb-000002
实施例8寡糖疫苗的免疫实验
利用免疫低下(中性粒细胞减少)小鼠模型来研究疫苗的有效性，这种中性粒血球减少小鼠模型被认为是合适的模型，可以用来模拟由于治疗各种恶性肿瘤化疗等而出现这种缺陷的患者。注射环磷酰胺导致的小鼠免疫受损会增加对真菌的敏感性，这与中性粒细胞减少症患者对该病的易感性增强相似。考察T细胞介导的免疫应答，取小鼠血液检测Th17细胞中细胞因子IL-17F水平的变化来评价壳寡糖-载体蛋白疫苗的免疫原性。
1、免疫低下小鼠模型的建立
选取6-8周龄Balb/c雌性小鼠，适应性喂养3-7天。用生理盐水配制20g/L的环磷酰胺，按200mg/kg体重腹腔注射环磷酰胺，3天后用SYSMEX XT-2000i全自动血液分析仪测小鼠血液中性粒细胞数量，中性粒细胞数量＜500个/mm 3，可继续实验，之后每10天注射一次环磷酰胺，剂量为150mg/kg，并维持整个试验期间。
2、小鼠免疫实验方案
设置生理盐水空白对照组、KLH组、壳寡糖组、实施例6中壳寡糖-KLH组、氟康唑组(作为攻毒试验阳性对照)，每组10只。在实验开始第0、2、4、6周，对小鼠颈部、背部进行皮下多点注射，注射剂量为2.5mg/kg，生理盐水空白组注射生理盐水，氟康唑组不作注射，在第6周按50mg/kg·d给小鼠灌胃，连续灌胃7天，其他组别注射含佐剂的受试样品。
为刺激机体产生较强的免疫反应，选用弗氏佐剂。第0周免疫注射选用弗氏完全佐剂，第2、4、6周免疫注射选用弗氏不完全佐剂。用0.02M PBS(pH7.2-7.4)缓冲液将KLH、壳寡糖、壳寡糖-KLH配制成1mg/mL溶液，再与弗氏完全佐剂或弗氏不完全佐剂等体积混合，通过超声波乳化均匀。
3、细胞因子检测
细胞因子测定步骤：
(1)淋巴细胞的提取和刺激：在第0周免疫注射前和第6周免疫注射后，对小鼠进行颌下静脉取血0.3mL于1.5mL的抗凝管混匀，加入3mL 1×红细胞裂解液，混匀反应4min，4℃ 500g离心10min，吸弃上清，再加入10mL 0.01M PBS后500g离心10min，吸弃上清，用200μL刺激液，将沉淀的淋巴细胞悬浮于圆底96孔板中，在细胞培养箱中孵育4h。
(2)胞外染色：将96孔板在1600rpm离心10min(离心条件下同)，弃上清，按70μL/孔添加含胞外染色抗体的FACS缓冲液(含荧光染色CD3e、CCR6、CD4、7-AAD抗体，添加量分别为1∶100、1∶100、1∶200、1∶100)吹打，避光孵育40min，进行胞外染色。
(3)破膜：加入不含抗体的FACS缓冲液180μL/孔并吹打，离心弃上清，按100μL/孔加入破膜液，避光4℃孵育40min，进行破膜处理。
(4)胞内染色：用1×的破膜缓冲液180μL/孔吹打，离心弃上清，按70μL/孔加入含胞内染色抗体的破膜缓冲液，室温避光孵育40min，进行胞内细胞因子染色。
(5)细胞因子检测：按180μL/孔添加FACS缓冲液并吹打，离心弃上清，再按100μL/孔加入FACS缓冲液并吹打，流式细胞仪检测Th17胞内IL-17F水平，反映受试样品对小鼠细胞免疫的激活作用。各组别小鼠免疫前后胞内IL-17F占Th17比例如表3所示。
表3小鼠免疫前后Th17胞内IL-17F占比(
Figure PCTCN2021000153-appb-000003
n＝10)
Figure PCTCN2021000153-appb-000004
注： ＊＊P＜0.01vs免疫前， ##P＜0.01vs空白组
由表3可见，免疫后与免疫前相比，KLH组、壳寡糖组小鼠Th17胞内IL-17F占比有所提高，但效果不显著，实施例6中的壳寡糖-KLH组小鼠胞内IL-17F占比明显提高(P＜0.01)；与空白对照组相比，KLH组、壳寡糖组胞内IL-17F占比有一定程度的提高，实施例6中的壳寡糖-KLH组小鼠胞内IL-17F占比有极显著提高(P＜0.01)，表明偶联有载体蛋白的寡糖能激起Th17细胞免疫，促使其分泌细胞因子IL-17F，起到抗真菌的作用。
实施例9免疫小鼠攻毒试验
以白色念珠菌作为攻毒菌株，研究寡糖疫苗对真菌攻毒后的保护作用。
1、白色念珠菌感染致死剂量的确定
用0.01M PBS将培养的白色念珠菌制成悬浮液，调整浓度为0.5×10 6、1.0×10 6、0.5×10 7、1.0×10 7、0.5×10 8、1.0×10 8CFU/ml，按剂量0.1ml/只经小鼠尾静脉注射攻毒，小鼠选取实施例8步骤1中注射环磷酰胺的免疫低下小鼠(中性粒细胞数量＜500个/mm 3)，同时设置生理盐水对照组，统计5天内小鼠死亡率，确定最小致死剂量，结果如表4所示。
表4小鼠尾静脉注射白色念珠菌致死率
Figure PCTCN2021000153-appb-000005
从表4可以看出，对照组小鼠未有死亡。0.5×10 5CFU和1.0×10 5CFU剂量组5天内小鼠未有死亡；0.5×10 6CFU剂量组5天内死亡率30％；0.5×10 7CFU剂量组5天死亡率达到100％；1.0×10 7CFU剂量组死亡率12h内死亡率为100％，可以看出，白色念珠菌对小鼠的最小致死剂量为0.5×10 7CFU。
2、免疫小鼠攻毒试验
同实施例8的免疫方案，第8周将致死剂量的白色念珠菌尾静脉注射攻毒，观察各组小鼠的死亡情况。7天后计算小鼠生存率，如表5所示。
表5免疫小鼠攻毒后存活率
Figure PCTCN2021000153-appb-000006
从上表可以看出，壳寡糖-KLH组小鼠存活率达到65％，明显高于其他组，说明制备的寡糖疫苗对白色念珠菌引起的感染具有免疫保护作用。
最后应该说明的是，以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，所属领域技术人员应该明白，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。

Claims (10)

  1. 一种特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，包括：
    对含有伯氨基的载体蛋白进行巯基化修饰，得到巯基化蛋白；
    将所述巯基化蛋白与壳寡糖混合物和/或几丁质寡糖混合物偶联，形成寡糖疫苗。
  2. 如权利要求1所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，所述壳寡糖混合物和/或几丁质寡糖混合物，优选的，壳寡糖混合物和/或几丁质寡糖的相对分子质量≤5000Da，脱乙酰度为0-100％。
  3. 如权利要求1所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，所述载体蛋白为非人源蛋白。
  4. 如权利要求3所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，所述非人源蛋白为Concholepasconcholepas hemocyanin、钥孔血蓝蛋白、牛血清白蛋白、破伤风毒素/类毒素、轮状病毒VP7蛋白、白喉毒素突变体CRM、产气荚膜羧状芽胞杆菌外毒素/类毒素中的一种。
  5. 如权利要求1所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，偶联在桥联剂存在条件下进行。
  6. 如权利要求5所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，所述桥联剂为3-(2-吡啶基二硫代)丙酰肼。
  7. 如权利要求1所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，偶联的具体步骤包括：
    向巯基化蛋白溶液中加入桥联剂，室温下进行反应；
    再加入壳寡糖混合物和/或几丁质寡糖混合物，4℃下反应；
    去除杂质小分子，得寡糖疫苗。
  8. 如权利要求1或7所述的特异性预防真菌感染的寡糖疫苗及其制备方法，其特征在于，壳寡糖混合物和/或几丁质寡糖混合物的摩尔浓度相当于巯基化蛋白摩尔量的100-1200倍；或，去除杂质小分子的方法为透析或柱层析。
  9. 权利要求1-8任一项所述的方法制备的特异性预防真菌感染的寡糖疫苗。
  10. 权利要求9所述的特异性预防真菌感染的寡糖疫苗在制备治疗或预防系统性真菌感染的药物、或激活Th17细胞免疫应答的药物中的应用。
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