Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli
Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli
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DOI：
10.1093/glycob/cwv077
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发表时间：
2016-01-01
期刊：
影响因子：
4.3
通讯作者：
Wacker, Michael
中科院分区：
文献类型：
--
作者：
Ravenscroft, Neil;Haeuptle, Micha A.;Wacker, Michael
志贺氏菌病仍然是发展中国家腹泻病的主要原因，并在儿童中造成相当大的发病率和死亡率。由细菌表面多糖与载体蛋白结合而成的糖结合疫苗是控制侵袭性细菌感染的最有效疫苗。然而，复杂的生产工艺阻碍了预防志贺氏菌病的多价结合疫苗的开发，因为每个菌株的表面多糖需要提取、水解、化学激活和与载体蛋白的结合。一种创新的生物合成大肠杆菌糖基化系统的使用大大简化了糖结合物的生产。在此，我们表达了志贺氏菌1型(SD1)O-多糖，并通过高效液相分析和质谱分析证明了其在大肠杆菌糖基载体脂质上的功能组装。该多糖通过PgIB寡糖转移酶和来自铜绿假单胞菌的载体蛋白外毒素A(EPA)的共表达而酶促连接到载体蛋白的特定天冬酰胺残基。本文报道了志贺氏菌糖偶联物(SD1-EPA)的提取和纯化，并用核磁共振和质谱学等物理化学方法对其进行了详细的表征。该报告首次表明，生物偶联提供了一种新开发和改进的方法来生产SD1糖偶联物，可以使用最先进的技术对其进行表征。此外，这种通用工艺和分析方法非常适合生产更多的志贺氏菌血清型，从而可以开发多价志贺氏菌疫苗。
Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PgIB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.