来自酿酒酵母的β-葡聚糖的佐剂特性研究

E. Volosnikova, D. N. Shcherbakov, N. V. Volkova, T. I. Esina, A. V. Zaikovskaya, G. G. Shimina, E. D. Danilenko
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引用次数: 0

摘要

为了提高现代疫苗(尤其是亚单位疫苗)的有效性和免疫原性,需要使用佐剂。多糖具有安全性和生物相容性,是制作疫苗佐剂的理想候选物质。我们的研究旨在开发一种从酵母细胞壁中获取β-葡聚糖的方法,并评估其佐剂特性。通过在纯化阶段使用纤维素酶和蛋白酶的酶复合物并结合超声波(22 千赫),制备出了高纯度和无毒性的制剂。根据所开发的方案,从 100 克湿细胞生物质中可获得高达 2 克的β-葡聚糖。对 50 只体重为 16-18 克的雄性 BALB/c 小鼠进行了β-葡聚糖佐剂特性的研究。免疫接种两次,每次间隔 14 天,每只小鼠肌肉注射 200 μl。以 SARS-CoV-2 病毒(武汉-Hu-1 和 B.1.617.2(Delta))表面 S 蛋白的重组受体结合域(RBD)为抗原,每只动物剂量为 50 微克。阳性对照组注射抗原和氢氧化铝。阴性对照组为注射生理盐水的小鼠。血清中特异性抗体的滴度通过 ELISA 方法测定。以 RBD(武汉-胡-1 和 Delta)和 S 蛋白(武汉-胡-1、Delta 和 Omicron)为抗原。用 SARS-CoV-2 病毒株武汉-胡-1、Delta(B.1.617.2)和 Omicron(B.1.1.529)进行中和试验,测定病毒中和抗体的滴度。研究结果表明,β-葡聚糖能提高用 RBD 免疫的小鼠产生特异性抗体和病毒中和抗体的能力。特异性抗体和病毒中和抗体的滴度与用 RBD 和 Al(OH)₃ 免疫组的水平相当。在白色近交系 ICR 小鼠的实验中发现,该制剂属于几乎无毒的物质。因此,可以得出结论:β-葡聚糖是一种生物相容性好、可生物降解、无毒、生产劳动密集型低的物质,可以替代传统的铝盐佐剂。
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Study of the adjuvant properties of beta-glucans from Saccharomyces Cerevisiae yeast
To increase the effectiveness and immunogenicity of modern vaccines, especially subunit ones, it is required to use adjuvants. Polysaccharides, due to their safety and biocompatibility, are desirable candidates for the creation of vaccine adjuvants. The aim of our study was to develop a method for obtaining beta-Glucans from the yeast Saccharomyces cerevisiae cell wall, and evaluate their adjuvant properties. The high purity and non-toxicity of the resulting preparation was achieved by using enzyme complexes of cellulase and protease in combination with ultrasound (22 kHz) at the purification stage. The developed scheme allows for the yield of beta-Glucans up to 2 g from 100 g of the biomass of wet cells. The adjuvant properties of beta-Glucans were studied in 50 male BALB/c mice, weighing 16–18 g. Immunization was performed twice, with a 14-day interval, intramuscularly, 200 μl per animal. The recombinant receptor-binding domain (RBD) of the surface S protein of the SARS-CoV-2 virus (Wuhan-Hu-1 and B.1.617.2 (Delta)) was used as an antigen, at a dose of 50 μg per animal. A positive control group was administered with the antigen combined with aluminum hydroxide. As a negative control, mice injected with the saline solution were used. The titers of specific antibodies in the blood sera were determined by ELISA assays. RBD (Wuhan-Hu-1 and Delta), and S protein (Wuhan-Hu-1, Delta and Omicron) were used as antigens. The titers of virus-neutralizing antibodies were measured in neutralization tests using SARS-CoV-2 virus strains Wuhan-Hu-1, Delta (B.1.617.2) and Omicron (B.1.1.529). The results of the study have shown that beta-Glucans have the ability to enhance the production of specific and virus-neutralizing antibodies in mice immunized with RBD. The titers of specific and virus neutralizing antibodies are comparable to their levels in the group immunized with RBD and Al(OH)₃. It has been found in the experiments in white outbred ICR mice that the preparation belongs to practically non-toxic substances. Therefore, it can be concluded that the use of beta-Glucans could become a preferable alternative to the conventional adjuvants based on aluminum salts, being biocompatible, biodegradable and non-toxic substances of low labor-intensive production.
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