Research and Development of a Novel System to Produce Polysaccharide Conjugate Va

多糖复合物生产新系统的研究与开发

• 批准号: 7673238
• 负责人: Peng George Wang
• 金额: $ 10.58万
• 依托单位: CARBOGENE USA, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7673238
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Bacterial Vaccines; Biochemical; Biological Assay; Carrier Proteins; Chemicals; Clinical; Communicable Diseases; Complex; Conjugate Vaccines; Development; Diphosphates; Disease; Engineering; Escherichia coli; Escherichia coli K12; Fermentation; Gene Cluster; Generations; Genes; Goals; Health; Immune response; In Vitro; Investigation; Ligation; Link; Lipid A; Lipids; Methods; Modeling; Monosaccharides; Pathogenicity; Pathway interactions; Peptides; Phase; Polysaccharides; Process; Production; Proteins; Reaction; Resistance; Secondary Protein Structure; Serotyping; Site; Small Business Technology Transfer Research; Streptococcus pneumoniae; Substrate Specificity; System; Thick; Vaccine Production; Vaccines; Variant; base; capsule; chemical genetics; chemical synthesis; cost; cross reacting material 197; fighting; glycosylation; in vivo; mutant; novel; pathogen; preference; prevent; public health relevance; research and development; research study; success; sugar; vaccine development

DESCRIPTION (provided by applicant): Polysaccharide conjugate vaccines are proving to be an effective means to generate protective immune responses so as to prevent a wide range of diseases. 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 a recently established bacterial protein O-glycosylation system to obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This bacterial glycosylation system includes a highly promiscuous protein, PglL, which catalyzes the transfer of polysaccharides from a diphospho-lipid donor to target proteins. The proof-of-concept experiments led compelling evidence for further exploration of such a system in conjugate vaccine development. Furthermore, the success of this proposed approach 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. Phase I of this STTR application will demonstrate the feasibility of this approach using a pneumococcal pathogen (Streptococcus pneumoniae serotype 14) as a model. We plan to focus our efforts on the following two specific aims: 1. We will establish an in vitro reaction system based on the chemical synthesis of lipid pyrophosphate linked monosaccharides and enzymatic assembly of the corresponding CPS repeating unit. Besides confirmation of PglL activity towards this specific CPS (CPS14), the peptide preference in the context of both the primary sequence and local secondary structure of proteins will be explored to obtain the most favorable substrate sequence for PglL. Finally, a commonly used carrier protein of polysaccharide conjugate vaccines (CRM197) will be engineered to serve as the acceptor substrate for an in vivo production system. 2. We aim to achieve an E. coli strain producing CRM197 conjugated CPS14. E. coli K12 W3110 will be utilized as a host strain. Two genes (wecA: initial glycosyltransfer reaction, waaL: ligation of nascent polysaccharide chains to the core-Lipid A) which are vital to the LPS biosynthetic pathway and may interfere with PglL O-glycosylation will be disrupted. The feasibility of heterologously expressing of CPS14 in the host strain will then be demonstrated. 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. Expression, purification, characterization and bioassays of CPS14-CRM197 conjugates from fermentation of this engineered strain will be explored. Successful demonstration of the production of CRM197 conjugated CPS14 will set the groundwork for producing CPS conjugates from other S. pneumoniae serotypes as well as polysaccharides from other pathogens. Given that the CPS biosynthetic gene clusters of 90 S. pneumoniae serotypes have been completely sequenced, 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 a single pathogen as a model. Then the established method can be easily applied to other pathogens.

描述（由申请方提供）：多糖缀合物疫苗被证明是产生保护性免疫应答以预防多种疾病的有效手段。传统的化学生产方法已经能够生产出几种非常成功的临床使用的结合疫苗。然而，传统的方法遭受多个发酵、纯化步骤、低产量和非特异性化学缀合，从而导致疫苗生产的高成本。本申请的目的是探索最近建立的细菌蛋白O-糖基化系统，以简便、有效和易于应用的方式获得多糖缀合物疫苗。该细菌糖基化系统包括高度混杂的蛋白质PglL，其催化多糖从二磷酸脂质供体转移到靶蛋白。概念验证实验为在缀合物疫苗开发中进一步探索这种系统提供了令人信服的证据。此外，这种方法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，我们在过去几年中一直在结合遗传，化学和生物化学方法进行研究。该STTR应用的第一阶段将使用肺炎球菌病原体（肺炎链球菌血清型14）作为模型来证明这种方法的可行性。我们计划集中努力实现以下两个具体目标：1.我们将建立一个基于脂焦磷酸连接单糖的化学合成和相应CPS重复单元的酶促组装的体外反应体系。除了确认PglL对该特异性CPS（CPS 14）的活性之外，还将探索在蛋白质的一级序列和局部二级结构的背景下的肽偏好，以获得PglL的最有利的底物序列。最后，多糖缀合物疫苗（CRM 197）的常用载体蛋白将被工程化以用作体内生产系统的受体底物。2.我们的目标是实现一个E。产生CRM 197缀合的CPS 14的大肠杆菌菌株。E. coli K12 W3110作为宿主菌。两个基因（wecA：初始糖基转移反应，waaL：新生多糖链与核心-脂质A的连接），这对LPS生物合成途径至关重要，并且可能干扰PglL O-糖基化将被破坏。然后将证明在宿主菌株中异源表达CPS 14的可行性。将编码PglL和有利的CRM 197变体的基因以及异源表达CPS 14所需的CPS 14基因簇沿着引入突变宿主菌株中。将探索来自该工程菌株发酵的CPS 14-CRM 197缀合物的表达、纯化、表征和生物测定。CRM 197结合CPS 14的生产的成功证明将为从其他链球菌生产CPS结合物奠定基础。肺炎血清型以及来自其他病原体的多糖。鉴于90 S.尽管已经对肺炎链球菌血清型进行了完全测序，但多糖生物合成基因座的可及性将使得我们的方法能够推广用于生产各种多糖缀合物疫苗。公共卫生相关性：细菌感染是全球主要的健康问题之一。多糖，形成包围细菌病原体的厚胶囊，代表致病性的主要决定因素。随着对主要抗生素耐药性的增加，开发基于多糖的疫苗为对抗感染性疾病提供了一种有吸引力的方法。多糖最好与载体蛋白结合作为结合疫苗以增强其效力。多糖缀合物疫苗生产的传统化学方法使得能够生产目前临床使用的几种非常成功的缀合疫苗。然而，传统的方法存在生产步骤复杂、产率低和产品不纯的缺点，从而导致疫苗生产成本高。本申请的目的是开发一种生产多糖结合疫苗的新方法。用这种方法，我们可以获得多糖结合疫苗在一个简单的，有效的，易于应用的方式。该方法将首先以单一病原体为模型进行探索。所建立的方法也可方便地应用于其他病原体的检测。