Development of A Novel Strategy to Produce Antibacterial Glycoconjugate Vaccines

开发生产抗菌糖复合物疫苗的新策略

• 批准号: 7932881
• 负责人: Peng George Wang
• 金额: $ 37.29万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932881
• 关键词: Adult; Anabolism; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Bacterial Vaccines; Biochemical; Biological Assay; Carbohydrates; Carrier Proteins; Chemicals; Child; Clinical; Communicable Diseases; Complex; Conjugate Vaccines; Coupled; Development; Diphosphates; Engineering; Enzymes; Escherichia coli; Escherichia coli K12; Escherichia coli O157; Fermentation; Gene Cluster; Generations; Genes; Glycoconjugates; Goals; Health; Hexoses; In Vitro; Infection prevention; Investigation; Knowledge; Length; Ligation; Link; Lipid A; Lipids; Mediating; Methods; Modeling; Monosaccharides; O Antigens; One-Step dentin bonding system; Pathogenicity; Peptides; Persons; Polysaccharides; Production; Protein Glycosylation; Proteins; Quality Control; Reaction; Regulation; Research; Resistance; Series; Streptococcus pneumoniae; System; Techniques; Thick; Vaccine Production; Vaccines; Variant; base; capsule; chemical genetics; chemical synthesis; cost; cross reacting material 197; fighting; glycosylation; high risk; immunogenic; immunogenicity; in vivo; interest; mouse model; mutant; novel; novel strategies; pathogen; periplasm; physical property; polymerization; preference; public health relevance; reconstitution; research study; success; sugar; vaccine development

DESCRIPTION (provided by applicant): Bacterial infections constitute one of the major health problems worldwide. A gradual increase in the resistance to antibiotics leads to a serious thread for successful treatment of bacterial infections. This feature has stimulated the interest in vaccines to prevent infections. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. Therefore, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. In contrast to pure polysaccharides, carbohydrates coupled to an immunogenic protein present enhanced immunogenicity. Glycoconjugate vaccine provides a long lasting protection for adults as well as for persons at high risk and young children. The traditional chemical producing approach has enabled the production of several highly successful conjugated vaccines in clinical use. However, the traditional approach suffers from multiple fermentations, purification steps, low yields and non-specific chemical conjugation, thus leading to high costs for vaccine production. The objective of this application is to explore two recently established bacterial protein glycosylation systems to obtain glycoconjugate vaccines in a facile, efficient, and easily applicable manner. These bacterial glycosylation systems comprise two highly promiscuous proteins (PglB for N-glycosylation and PglL for O-glycosylation), which catalyze the transfer of polysaccharides from a diphospho-lipid donor to target proteins. The proof-of-concept experiments conducted by our lab and other groups lend compelling evidence for further exploration of such a system in the glyconjugate vaccine development. Furthermore, the success of this proposed research also heavily relies on the in-depth understanding of polysaccharide biosynthesis in bacteria, which we have been studying in the past several years with a combination of genetic, chemical, and biochemical approaches. To fulfill this objective, we proposed three specific aims: Aim 1. Investigation of PglB catalyzed N-glycosylation system The glycoconjugate synthesis of E. coli O157:H7 O-antigen attached to a commonly used carrier protein (CRM197) by PglB involved in N-glycosylation system will be demonstrate in vitro and in vivo. Based on the chemical synthesis of lipid pyrophosphate linked monosaccharides, enzymatic assembly of the corresponding O-antigen repeating unit, reconstitution of O-antigen polymerization, and over-expression and purification of PglB and CRM197, an in vitro reaction system will be established. With this system, CRM197 will be engineered to be a PglB acceptor substrate and certain scale of glycoconjugates will be obtained for characterization. Then development of an in vivo fermentation system that contains all the necessary components for glycoconjugate production will be explored. E. coli K12 W3110 will be utilized as a host strain. Gene waaL responsible for ligation of nascent polysaccharide chains to the core-Lipid A will be disrupted to accumulate O-antigen chains in the periplasm. Furthermore, E. coli O157 LPS biosynthesis gene cluster will be introduced into E. coli K12 W3110 waaL strain. Finally, CRM197 variants obtained from in vitro study will be co-expressed with PglB in this engineered E. coli model strain to produce O- antigen-CRM197 glycoconjugate. Regulation of O-antigen chain length will also be explored. Aim 2. Investigation of PglL catalyzed O-glycosylation system PglB only tolerates glycan donor with a C2 acetamido group at the reducing end sugar while PglL was showed to be able to transfer glycan donor with hexose at the reducing end. Since the majority of CPS glycans contain hexose at the reducing end, PglL has higher potential for the generation of CPS conjugate vaccines than PglB. We choose S. pneumoniae type 14 as a model to demonstrate PglL mediated glycoconjugate vaccine production. The repeating unit of CPS from this strain (CPS14) will be reconstructed in vitro and used as sugar donor for in vitro reaction. The importance of lipid moiety for PglL recognition will be investigated. The substrate requirement and peptide preference for PglL protein acceptor will be determined and the commonly used carrier protein CRM197 will be engineered into a PglL substrate with the knowledge obtained from these studies. Then the feasibility of heterologously expressing of CPS14 in the host strain will then be investigated. Genes encoding PglL and the favorable CRM197 variants, along with the CPS14 gene cluster required for the heterologous expression of CPS14, will be introduced into the mutant host strain. Production and purification of CPS14-CRM197 conjugates from fermentation of this engineered strain will be explored. Aim 3. Quality control and bioactivity assay of polysaccharide conjugated vaccines A series of techniques including NMR, MS, CD will be employed to characterize the glycoconjugate vaccines produced in our systems. Bioactivity of the vaccines will be further evaluated in mice models. Successful demonstration of the production of CRM197 conjugated with E. coli O157:H7 O-antigen and S. pneumoniae type14 CPS will set the groundwork for producing glyconjugate vaccine for other pathogens. The accessibility of polysaccharide biosynthesis loci will enable generalization of our approach for the production of a variety of polysaccharide conjugate vaccines. PUBLIC HEALTH RELEVANCE: Bacterial infections are one of the major health problems worldwide. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. With the increasing emergence of resistance toward major antibiotics, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. Polysaccharides are better to be conjugated to a carrier protein as conjugate vaccines to enhance their efficacy. The traditional chemical approach of polysaccharide conjugate vaccine production has enabled the production of several highly successful conjugated vaccines currently in clinical use. However, the traditional method suffers from complex production steps, low yields and impure products, thus leading to high costs for vaccine production. The objective of this application is to develop a novel method for polysaccharide conjugate vaccine production. With this method, we can obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This method will firstly be explored with model pathogens. Then the established method can be easily applied to other pathogens.

描述（由申请人提供）：细菌感染是全球主要的健康问题之一。对抗生素耐药性的逐渐增加导致成功治疗细菌感染的严重问题。这一特点激发了人们对预防感染的疫苗的兴趣。在细菌病原体周围形成厚囊的多糖是致病性的主要决定因素。因此，开发以多糖为基础的疫苗为防治传染病提供了一条有吸引力的途径。与纯多糖相比，与免疫原性蛋白偶联的碳水化合物具有增强的免疫原性。糖结合疫苗为成人以及高危人群和幼儿提供了持久的保护。传统的化学生产方法已经能够生产几种临床使用的非常成功的结合疫苗。然而，传统方法存在多个发酵、纯化步骤、产量低和非特异性化学偶联的问题，从而导致疫苗生产成本高。本应用的目的是探索两种最近建立的细菌蛋白糖基化系统，以一种简单、有效和易于应用的方式获得糖结合疫苗。这些细菌糖基化系统包括两个高度混杂的蛋白质（n -糖基化PglB和o-糖基化PglL），它们催化多糖从二磷酸脂供体转移到靶蛋白。我们的实验室和其他团队进行的概念验证实验为进一步探索糖偶联疫苗开发中的这种系统提供了令人信服的证据。此外，这项研究的成功也在很大程度上依赖于对细菌中多糖生物合成的深入了解，这是我们在过去几年中结合遗传，化学和生化方法进行的研究。为实现这一目标，我们提出了三个具体目标：本文将在体外和体内验证PglB参与n -糖基化系统对大肠杆菌O157:H7 o -抗原与常用载体蛋白（CRM197）结合的糖缀合反应。基于脂质焦磷酸连接单糖的化学合成、相应o抗原重复单元的酶组装、o抗原聚合重构、PglB和CRM197的过表达纯化，建立体外反应体系。利用该系统，CRM197将被设计成PglB受体底物，并获得一定规模的糖缀合物进行表征。然后开发一种体内发酵系统，其中包含所有必要的成分糖缀合物的生产将被探索。大肠杆菌K12 W3110将被用作宿主菌株。负责将新生多糖链连接到核心脂质A的基因waaL将被破坏，从而在周围质中积累o抗原链。进一步，将大肠杆菌O157脂多糖生物合成基因簇导入大肠杆菌K12 W3110 waaL菌株。最后，从体外研究中获得的CRM197变异体将在该工程大肠杆菌模型菌株中与PglB共表达，以产生O-抗原-CRM197糖缀合物。对o型抗原链长度的调控也将进行探讨。目标2。pgl催化o糖基化体系的研究pgl只耐受在还原端有C2乙酰氨基的糖供体，而pgl能够转移在还原端有己糖的糖供体。由于大多数CPS聚糖在还原端含有己糖，因此PglL比PglB更有可能生成CPS结合疫苗。我们选择肺炎链球菌14型作为模型来证明pgl介导的糖结合疫苗的生产。该菌株的CPS重复单元（CPS14）将在体外重建，并作为糖供体进行体外反应。脂质部分对pgl识别的重要性将被研究。将确定pll蛋白受体的底物需求和肽偏好，并利用从这些研究中获得的知识将常用的载体蛋白CRM197改造成pll底物。然后研究CPS14在宿主菌株中异源表达的可行性。编码pgl和有利的CRM197变异体的基因，以及CPS14异源表达所需的CPS14基因簇，将被引入突变宿主菌株。该工程菌株发酵过程中CPS14-CRM197偶联物的生产和纯化将进行探索。目标3。多糖结合疫苗的质量控制和生物活性测定将采用核磁共振、质谱、CD等一系列技术对我们系统生产的多糖结合疫苗进行表征。疫苗的生物活性将在小鼠模型中进一步评估。成功生产与大肠杆菌O157:H7 o抗原和肺炎链球菌14型CPS结合的CRM197将为生产针对其他病原体的糖结合疫苗奠定基础。多糖生物合成位点的可及性将使我们的方法推广到生产各种多糖结合疫苗。