WO2022226672A1 - 一种预防真菌感染的几丁质寡糖疫苗及其制备方法 - Google Patents

一种预防真菌感染的几丁质寡糖疫苗及其制备方法 Download PDF

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WO2022226672A1
WO2022226672A1 PCT/CN2021/000154 CN2021000154W WO2022226672A1 WO 2022226672 A1 WO2022226672 A1 WO 2022226672A1 CN 2021000154 W CN2021000154 W CN 2021000154W WO 2022226672 A1 WO2022226672 A1 WO 2022226672A1
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vaccine
oligosaccharide
fungal infection
chitin
chitin oligosaccharide
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PCT/CN2021/000154
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English (en)
French (fr)
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袁丹丹
刘飞
陈磊
牛林林
陈勉
马志平
张艳艳
袁超
张秀华
张晓元
张林军
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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 a chitin oligosaccharide vaccine for preventing fungal infection and a preparation method thereof.
  • Fungal cell walls generally contain mannose, ⁇ -glucan and chitin polysaccharides. Previous studies have found that sugars have low immunogenicity and are difficult to stimulate the immune effect of the body. Some research teams have coupled ⁇ -mannobiose, trisaccharides, and tetrasaccharides with keyhole limpet hemocyanin (KLH) to form glycoprotein compounds.
  • KLH keyhole limpet hemocyanin
  • oligosaccharide vaccine to prevent invasive fungal infection, which is achieved by combining the amino group (-NH 2 ) on the chitosan oligosaccharide with the amino group (-NH 2 ) on the carrier protein, through a bridging agent (such as CHO (CH 2 ). 2 )
  • a bridging agent such as CHO (CH 2 ). 2
  • the coupling effect of mCHO ) to form glycoprotein compounds which can effectively activate Th17 and Th1 cell immunity, but because chitosan oligosaccharide contains multiple amino groups, the coupling position with the protein is not clear, the specificity is poor, the process is unstable, and the quality Can not control.
  • Chitin is a component widely present in fungal cell walls.
  • Chitin oligosaccharide is a small molecule oligosaccharide obtained by enzymatic hydrolysis of chitin. It has the same monosaccharide composition as chitin and can be used as a broad-spectrum antifungal agent. target.
  • a series of chitin oligosaccharides have been prepared in vitro in batches, enabling large-scale immunogen screening.
  • no broad-spectrum fungal vaccine has been reported in this field, and the mechanism of its recognition by the immune system is still unclear. Therefore, the research of antifungal vaccine based on fungal cell structure has very urgent practical significance.
  • the purpose of the present invention is to provide a chitin oligosaccharide vaccine for preventing fungal infection, for preventing and treating the infection caused by fungi in immunosuppressed patients.
  • a first aspect of the present invention provides a method for preparing a chitin oligosaccharide vaccine for preventing fungal infection, comprising:
  • the aldehyde group-containing oligosaccharide mixture is coupled with a carrier protein to form a conjugate vaccine.
  • the oligosaccharide used in the present invention is chitin oligosaccharide, which is a mixture, has different degrees of polymerization, and has not been chemically modified, has strong structural similarity with chitin in fungal cell walls, and is easier to Causes an immune response in the body's cells.
  • the aldehyde group (-CHO) contained in the chitin oligosaccharide and the amino group (-NH 2 ) contained in the carrier protein with immunostimulatory effect can form a coupling site, and the obtained conjugate vaccine is not only structurally stable, but also The conjugation site is clear and specific, and it can well prevent and treat infections caused by fungi in immunosuppressed patients.
  • the second aspect of the present invention provides a chitin oligosaccharide vaccine for preventing fungal infection prepared by any of the above methods.
  • the third aspect of the present invention provides the application of the above-mentioned chitin oligosaccharide vaccine for preventing fungal infection in the preparation of a medicine for preventing systemic fungal infection.
  • the fourth aspect of the present invention provides the application of the above-mentioned chitin oligosaccharide vaccine for preventing fungal infection in preparing a medicine for activating Th17 cell immunity.
  • the oligosaccharide vaccine provided by the present invention utilizes the aldehyde group (-CHO) contained in chitin oligosaccharide to form a coupling site with the amino group (-NH 2 ) contained in the carrier protein with immunostimulatory effect, and adding reducing The agent forms a structurally stable conjugate vaccine.
  • the conjugated vaccine has a clear conjugation position and strong specificity.
  • the chitin oligosaccharide used is a mixture of different degrees of polymerization, which has strong structural components similar to chitin (ie polysaccharide) in the fungal cell wall.
  • the immunogenicity of the vaccine is improved, the Th17 cell immune response is effectively activated, and fungi (such as Candida albicans, Aspergillus, novel infection caused by Cryptococcus, etc.).
  • a chitin oligosaccharide vaccine for preventing fungal infection is formed by the condensation of an aldehyde group (-CHO) on an oligosaccharide mixture and an amino group (-NH 2 ) on a carrier protein to form a compound having a Schiff base ( Conjugation intermediate), the formation of Schiff base is a relatively unstable reversible interaction, which is converted into a stable conjugate under the action of reducing agent, that is, oligosaccharide vaccine, the coupling of oligosaccharide and carrier protein.
  • the combined molar ratio is (200-500): 1 or more.
  • the oligosaccharide is chitin oligosaccharide, which is a mixture of different degrees of polymerization, the relative molecular mass is less than or equal to 5000Da, and the degree of deacetylation is less than or equal to 15%, and the chitin oligosaccharide is commercially available or Prepared by degrading chitin.
  • the carrier protein is a non-human protein selected from Concholepasconcholepas hemocyanin (CCH), keyhole limpet hemocyanin (KLH, keyhole limpet hemocyanin), bovine serum albumin (BSA), tetanus toxin/ One of toxoid, rotavirus VP7 protein, diphtheria toxin mutant CRM, Bacillus perfringens exotoxin/toxoid.
  • CCH Concholepasconcholepas hemocyanin
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxin/ One of toxoid rotavirus VP7 protein
  • diphtheria toxin mutant CRM diphtheria toxin mutant CRM
  • Bacillus perfringens exotoxin/toxoid Bacillus perfringens exotoxin/toxoid.
  • the present invention also provides a method for preparing the above-mentioned chitin oligosaccharide vaccine for preventing fungal infection, comprising the following steps:
  • the concentration of carrier protein is 1-100 mg/ml
  • the phosphate buffer is 0.01-0.1M sodium phosphate, 0-0.15M sodium chloride, pH7.0-8.0 .
  • the molar concentration of chitin oligosaccharide is equivalent to 100-1200 times the molar amount of carrier protein.
  • the reducing agent is lithium aluminum hydride (LiAlH 4 ), potassium borohydride (KBH 4 ), sodium borohydride (NaBH 4 ), sodium cyanoborohydride (NaBH 3 CN), One of Na 2 SO 3 and FeSO 4 is added at a concentration of 1-100 mg/ml, and the reaction is placed at 25-37° C. for 1-10 days.
  • the purification method is column chromatography or dialysis for removing impurity small molecules
  • the buffer used for column chromatography or dialysis is the solution used for dissolving 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.
  • 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.
  • the small impurity molecules were removed through a dialysis bag with a molecular weight cut-off of 10000Da to obtain a chitin oligosaccharide-carrier protein conjugate, which was freeze-dried in vacuo. Oligosaccharide vaccine.
  • the small impurity molecules were removed by a dialysis bag with a molecular weight cut-off of 5000Da to obtain a chitin oligosaccharide-carrier protein conjugate, which was freeze-dried in vacuo. Oligosaccharide vaccine.
  • the small impurity molecules were removed through a dialysis bag with a molecular weight cut-off of 5000Da to obtain a chitin oligosaccharide-carrier protein conjugate, which was vacuum freeze-dried to obtain oligosaccharide.
  • Sugar vaccine 0.01M sodium phosphate and 0.15M sodium chloride with pH 8.0 as the buffer for dialysis
  • the small impurity molecules were removed through a dialysis bag with a molecular weight cut-off of 5000Da to obtain a chitin oligosaccharide-carrier protein conjugate, which was vacuum freeze-dried to obtain oligosaccharide.
  • Sugar vaccine 0.01M sodium phosphate and 0.15M sodium chloride with pH 8.0 as the buffer for dialysis
  • Example 7 Determination of chitin oligosaccharide-carrier protein coupling degree Taking Example 3 as an example, the coupling degree is represented by the coupling ratio, and the coupling ratio is the number of moles of chitin oligosaccharide connected to each mole of carrier protein.
  • Aspirate 30 ⁇ L of the standard or solution to be tested add 30 ⁇ L DNS, mix well, take a boiling water bath for 5 min, cool with tap water, add 180 ⁇ L purified water, mix well, add 200 ⁇ L to a 96-well plate, and measure the absorbance OD 540 with a microplate reader.
  • the concentration of reducing sugar in the sample and the chitin oligosaccharide solution was determined by the standard curve.
  • N-acetyl-D-glucosamine dissolve it in a 10ml volumetric flask with purified water, adjust the volume to the mark and shake well to obtain a 100mg/ml standard solution mother solution, which should be avoided in a 4°C refrigerator.
  • Light storage effective within two weeks. Pipette 0, 20, 40, 80, 120, 160, 200, 240, 280 ⁇ L N-acetyl-D-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.
  • 20 10 and 5 mg/ml chitin oligosaccharide solutions were prepared with purified water.
  • Pipette 100 ⁇ L of the standard or solution to be tested add 300 ⁇ L anthrone-sulfuric acid solution, take a boiling water bath for 10 min, immediately put it into ice water for 15 min, take 200 ⁇ L and put it into a 96-well plate, and measure the absorbance OD620 with a microplate reader.
  • the concentration of total sugar in the sample and chitin oligosaccharide solution was determined by the standard curve.
  • the degree of polymerization of chitin oligosaccharides the moles of total chitin oligosaccharides / the moles of chitin oligosaccharides reducing sugars;
  • the number of moles of chitin oligosaccharides attached to the protein (total sugar - reducing sugar ⁇ degree of polymerization) / degree of polymerization;
  • Coupling ratio moles of chitin 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 chitin oligosaccharide-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.
  • CCH, chitin oligosaccharide and chitin oligosaccharide-CCH were prepared into 1 mg/mL solution with 0.02M PBS (pH7.2-7.4) buffer, then mixed with Freund's adjuvant in equal volume, and homogeneously emulsified by ultrasonic .
  • 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.
  • 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 (neutrophil count ⁇ 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

一种预防真菌感染的几丁质寡糖疫苗及其制备方法，该疫苗是由寡糖与载体蛋白偶联而成，是通过寡糖上的醛基（-CHO）和载体蛋白上的氨基（-NH2）缩合并经过还原剂的作用而形成的偶联体。所述的载体蛋白为非人源蛋白，所述寡糖为几丁质寡糖的混合物。提供的疫苗免疫原性强，能够激活Th17细胞免疫，可以识别并保护真菌所致的感染。

Description

一种预防真菌感染的几丁质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，具体涉及一种预防真菌感染的几丁质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。
近年来，由于免疫缺陷或受损患者的不断增多(如免疫抑制剂使用、肿瘤放化疗、艾滋病流行、骨髓移植等)、胃肠道菌群失调患者的增多(如使用胃肠道复杂手术、广谱抗生素等)、侵入性置管患者的增多(如机械呼吸、深静脉营养等)，系统性真菌感染的发病率和死亡率在全球一直呈上升趋势。系统性真菌感染的常见致病株是念珠菌、曲霉菌、新型隐球菌，约占真菌感染的70％左右，感染累及人体内脏或形成真菌血症造成死亡。目前临床应用的抗真菌感染的药物品种少，这些抗真菌药物的大量使用，导致耐药菌株不断出现，产生了严重的耐药性问题。由于系统性真菌感染具有隐蔽性强、临床标本分离诊断的不确定性，在临床上没有明显表征，前期感染较难诊断，容易耽误最佳治病时机。真菌疫苗是最理想的预防措施，但真菌疫苗的研究起步晚、进展小，目前国内外仍无上市的预防系统性真菌感染的疫苗。
真菌细胞壁一般含甘露糖、β-葡聚糖及几丁质多糖。前期研究发现糖的免疫原性较低，难以激发机体的免疫效应，有研究团队将β-甘露二糖、三糖、四糖与钥孔血蓝蛋白(KLH)偶联形成糖蛋白化合物，并偶联白色念珠菌毒力蛋白Als1表位短肽，以及不同分支的β-葡聚糖与KLH偶联形成的糖蛋白化合物，均能在小鼠体内引起强烈的体液免疫应答(IgG抗体反应)，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。有研究提供了一种预防侵袭性真菌感染的寡糖疫苗，是通过壳寡糖上的氨基(-NH 2)与载体蛋白上的氨基(-NH 2)，通过桥联剂(如CHO(CH 2) mCHO)的联接作用，形成糖蛋白化合物，可有效激活Th17、Th1细胞免疫，但由于壳寡糖含有多个氨基，与蛋白的偶联位置不明确，特异性差，工艺不稳定，质量不可控。
几丁质是广泛存在于真菌细胞壁的成分，几丁质寡糖是几丁质经过酶解后得到的小分子寡糖，具有与几丁质相同的单糖组成，可作为广谱抗真菌的靶点。目前，已能在体外批量制备系列几丁质寡糖，可实现规模化免疫原筛选。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预防真菌感染的几丁质寡糖疫苗，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，提供了一种预防真菌感染的几丁质寡糖疫苗的制备方法，包括：
将含醛基的寡糖混合物与载体蛋白偶联，形成偶联体疫苗。
研究发现：本发明所用的寡糖为几丁质寡糖，其为混合物，具有不同的聚合度，且没有被化学修饰，与真菌细胞壁中的几丁质有很强的结构相似性，更容易引起机体细胞的免疫应答。同时，几丁质寡糖含有的醛基(-CHO)与具有免疫增活作用的载体蛋白含有的氨基(-NH 2)能够形成偶联位点，得到的偶联体疫苗不仅结构稳定，而且偶联位置明确，特异性强，能够很好地预防和治疗免疫抑制患者真菌引起的感染。
本发明的第二个方面，提供了任一上述的方法制备的预防真菌感染的几丁质寡糖疫苗。
本发明的第三个方面，提供了上述的预防真菌感染的几丁质寡糖疫苗在制备预防系统性真菌感染的药物中的应用。
本发明的第四个方面，提供了上述的预防真菌感染的几丁质寡糖疫苗在制备激活Th17细胞免疫的药物中的应用。
本发明的有益效果在于：
(1)本发明提供的寡糖疫苗，利用几丁质寡糖含有的醛基(-CHO)与具有免疫增活作用的载体蛋白含有的氨基(-NH 2)形成偶联位点，加入还原剂形成结构稳定的偶联体疫苗。偶联体疫苗的偶联位置明确，特异性强，其中，使用的几丁质寡糖为不同聚合度的混合物，与真菌细胞壁中的几丁质(即多聚糖)具有强的结构成分相似性，通过增加包括聚合度多样性，以及寡糖与载体蛋白的偶联比，提高了疫苗免疫原性，有效激活了Th17细胞免疫应答，识别并保护真菌(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。
一种预防真菌感染的几丁质寡糖疫苗，所述疫苗是是通过寡糖混合物上的醛基(-CHO)和载体蛋白上的氨基(-NH 2)缩合形成具有席夫碱类化合物(偶联中间体)，席夫碱的形成是一种相对不稳定的可逆相互作用，在还原剂的作用下将其转化为稳定的偶联体，即寡糖疫苗，寡糖与载体蛋白的偶联摩尔比达(200-500)∶1以上。
在一些实施例中，所述的寡糖为几丁质寡糖，其为不同聚合度的混合物，相对分子质量≤5000Da，脱乙酰度≤15％，所述几丁质寡糖为市售或通过降解几丁质制备。
在一些实施例中，所述的载体蛋白为非人源蛋白，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。
本发明还提供了上述预防真菌感染的几丁质寡糖疫苗的制备方法，包括如下步骤：
(1)将载体蛋白溶于磷酸盐缓冲液，颠倒混匀。
(2)加入几丁质寡糖，混匀。
(3)加入还原剂混匀，放置反应一定时间。
(4)纯化获得寡糖疫苗。
在一些实施例中，步骤(1)中，载体蛋白的浓度为1-100mg/ml，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。
在一些实施例中，步骤(2)中，几丁质寡糖的摩尔浓度相当于载体蛋白摩尔量的100-1200倍。
在一些实施例中，步骤(3)中，还原剂为氢化铝锂(LiAlH 4)、硼氢化钾(KBH 4)、硼氢化钠(NaBH 4)、氰基硼氢化钠(NaBH 3CN)、Na 2SO 3、FeSO 4中的一种，添加浓度为1-100mg/ml，25-37℃放置反应1-10天。
在一些实施例中，步骤(4)中，所述纯化方法为柱层析或透析，用于去除杂质小分子，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。
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
取80mg CCH溶于1ml pH7.2的0.01M磷酸钠、0.01M氯化钠缓冲液中，轻轻振荡溶解，加入2mg脱乙酰度为5％、分子量≤5000Da的几丁质寡糖，溶解混匀，16mg KBH 4，轻振混匀，25℃条件下放置反应2天。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，得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例2
取40mg CCH溶于1ml pH7.0的0.01M磷酸钠、0.01M氯化钠(pH7.2)缓冲液中，轻轻振荡溶解，加入4mg脱乙酰度为2％、分子量≤2000Da的几丁质寡糖，溶解混匀，40mg KBH 4，轻振混匀，28℃条件下放置反应3天。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5000Da透析袋除去杂质小分子，得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例3
取10mg CCH溶于1ml pH7.0的0.1M磷酸钠、0.01M氯化钠缓冲液中，轻轻振荡溶解，加入5mg脱乙酰度为1％、分子量≤1500Da的几丁质寡糖，溶解混匀，20mg KBH 4，轻振混匀，37℃条件下放置反应7天。以0.1M磷酸钠、0.01M氯化钠(pH7.0)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，经检测后得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例4
取10mg BSA溶于1ml pH7.0的0.01M磷酸钠缓冲液中，轻轻振荡溶解，加入25mg脱乙酰度为15％、分子量≤800Da的几丁质寡糖，溶解混匀，40mg NaBH 3CN，轻振混匀，25℃条件下放置反应1天。以pH7.0的0.01M磷酸钠缓冲液为流动相，通过Sephadex G10 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，经检测后得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例5
取50mg BSA溶于1ml pH8.0的0.01M磷酸钠、0.15M氯化钠缓冲液中，轻轻振荡溶解，加入75mg脱乙酰度为15％、分子量≤500Da的壳寡糖，溶解混匀，50mg LiAlH 4，轻振混匀，37℃条件下放置反应1天。以pH8.0的0.01M磷酸钠、0.15M氯化钠为透析用缓冲液，通过截流分子量5000Da透析袋除去杂质小分子，得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例6
取50mg破伤风毒素溶于1ml pH8.0的0.01M磷酸钠、0.15M氯化钠缓冲液中，轻轻振荡溶解，加入45mg脱乙酰度为15％的壳寡糖、分子量≤1000-2000Da，溶解混匀，10mg LiAlH 4，轻振混匀，37℃条件下放置反应1天。以pH8.0的0.01M磷酸钠、0.15M氯化钠为透析用缓冲液，通过截流分子量5000Da透析袋除去杂质小分子，得到几丁质寡糖-载体蛋白偶联体，真空冷冻干燥得寡糖疫苗。
实施例7几丁质寡糖-载体蛋白偶联程度测定以实施例3为例，偶联程度以偶联比表示，偶联比为每摩尔载体蛋白上连接的几丁质寡糖摩尔数。
(1)还原糖(游离几丁质寡糖)含量测定
称取N-乙酰-D-氨基葡萄糖0.1g(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10mg/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、20、40、80、120、160、240μLN-乙酰-D-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2、1、0.5mg/ml几丁质寡糖溶液。
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N-乙酰-D-氨基葡萄糖摩尔浓度为横坐标，吸光度为纵坐标，制定标准曲线，得标准曲线y＝148.18x-0.0585，R 2＝0.9991。以标准曲线测定样品及几丁质寡糖溶液中还原糖的浓度。
(2)总糖含量测定
称取N-乙酰-D-氨基葡萄糖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、20、40、80、120、160、200、240、280μLN-乙酰-D-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20、10、5mg/ml几丁质寡糖溶液。
吸取标准品或待测溶液100μL，加入300μL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OD620。以N-乙酰-D-氨基葡萄糖摩尔浓度为横坐标，吸光度为纵坐标，制定标准曲线，得标准曲线y＝8.9869x+0.0232，R 2＝0.9829。以标准曲线测定样品及几丁质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。
取标准溶液或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＝1.6331x+0.0761，R 2＝0.9884。以标准曲线测定样品中蛋白的浓度。
(4)偶联比的计算
几丁质寡糖的聚合度＝几丁质寡糖总糖摩尔数/几丁质寡糖还原糖摩尔数；
连在蛋白上的几丁质寡糖的摩尔数＝(总糖-还原糖×聚合度)/聚合度；
偶联比＝连在蛋白上的几丁质寡糖的摩尔数/(10×蛋白的摩尔数)。
(5)结果分析
通过还原糖测定发现，20管层析样品均未有颜色反应，说明无还原糖(游离几丁质寡糖)存在，即游离壳寡糖的上的醛基(-CHO)反应完全；总糖测定显示，第3、4、5管具有颜色反应，显棕色，吸光值较大，其它未显色；Bradford法测蛋白，第3、4、5管具有明显的颜色反应，显亮蓝色，吸光值较大，结果说明几丁质寡糖与载体蛋白发生偶联的层析部位集中在第3、4、5管，几丁质寡糖的聚合度计算如表1所示，第3、4、5管样品偶联比分别为321、3395、2302，数据如表2所示，第4管样品具有最高的偶联比，可作为小鼠免疫用制剂。
表1几丁质寡糖的聚合度
Figure PCTCN2021000154-appb-000001
Figure PCTCN2021000154-appb-000002
表2几丁质寡糖与载体蛋白的偶联数据
Figure PCTCN2021000154-appb-000003
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水平的变化来评价几丁质寡糖-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、CCH组、几丁质寡糖组、实施例3中几丁质寡糖-CCH组(第4管样品)、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CCH、几丁 质寡糖、几丁质寡糖-CCH配制成1mg/mL溶液，再与弗氏佐剂等体积混合，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3mL1×红细胞裂解液，混匀反应4min，4℃500g离心10min，吸弃上清，再加入10mL 0.01MPBS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 PCTCN2021000154-appb-000004
n＝10)
Figure PCTCN2021000154-appb-000005
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，CCH组、几丁质寡糖组小鼠Th17胞内IL-17F占比有所提高，但效果不显著，实施例3中的几丁质寡糖-CCH组小鼠胞内IL-17F占比明显提高(P＜0.01)；与空白对照组相比，CCH组、几丁质寡糖组胞内IL-17F占比有一定程度的提高， 实施例3中的几丁质寡糖-CCH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 PCTCN2021000154-appb-000006
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 PCTCN2021000154-appb-000007
Figure PCTCN2021000154-appb-000008
从上表可以看出，几丁质寡糖-CCH组小鼠存活率达到70％，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. 一种预防真菌感染的几丁质寡糖疫苗及其制备方法，其特征在于，包括：
    将含醛基的寡糖混合物与载体蛋白偶联，形成寡糖混合物偶联体疫苗。
  2. 如权利要求1所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，所述偶联包括：寡糖混合物上的醛基和载体蛋白上的氨基的缩合以及还原剂处理。
  3. 如权利要求1所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，所述的寡糖为具有不同聚合度的几丁质寡糖混合物，相对分子质量≤5000Da，脱乙酰度≤15％。
  4. 如权利要求1所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，所述的载体蛋白为非人源蛋白，优选的，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所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，还原剂为氢化铝锂、硼氢化钾、硼氢化钠、氰基硼氢化钠、Na 2SO 3、FeSO 4中的一种，优选的，浓度为1-100mg/ml。
  6. 如权利要求1所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，还包括：去除杂质小分子、冻干处理；优选的，去除杂质小分子的方法为透析或柱层析。
  7. 如权利要求1所述的预防真菌感染的几丁质寡糖疫苗的制备方法，其特征在于，所述的载体蛋白的浓度为1-100mg/ml；
    或，所述的几丁质寡糖的摩尔浓度相当于载体蛋白摩尔量的100-1200倍；
    或，加入还原剂后的放置反应时间为1-10天。
  8. 权利要求1-7任一项所述的方法制备的预防真菌感染的几丁质寡糖疫苗。
  9. 权利要求8所述的预防真菌感染的几丁质寡糖疫苗在制备预防系统性真菌感染的药物中的应用。
  10. 权利要求8所述的预防真菌感染的几丁质寡糖疫苗在制备激活Th17细胞免疫的药物中的应用。
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