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（由申请人提供）：多糖结合疫苗被证明是产生保护性免疫反应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在临床使用中非常成功的结合疫苗。然而，传统方法存在多次发酵、纯化步骤、产量低和非特异性化学结合等问题，从而导致疫苗生产成本高昂。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、高效且易于应用的方式获得多糖结合疫苗。这种细菌糖基化系统包括高度混杂的蛋白质 PglL，它催化多糖从二磷脂供体转移到目标蛋白质。概念验证实验为在结合疫苗开发中进一步探索此类系统提供了令人信服的证据。此外，这种方法的成功在很大程度上依赖于对细菌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、化学和生化方法进行研究。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）作为模型来证明该方法的可行性。我们计划重点致力于以下两个具体目标： 1. 我们将建立基于脂质焦磷酸连接单糖的化学合成和相应的CPS重复单元的酶促组装的体外反应体系。除了确认 PglL 对这种特定 CPS (CPS14) 的活性外，还将探索蛋白质一级序列和局部二级结构背景下的肽偏好，以获得 PglL 最有利的底物序列。最后，一种常用的多糖结合疫苗载体蛋白（CRM197）将被设计为体内生产系统的受体底物。 2. 我们的目标是获得产生 CRM197 缀合 CPS14 的大肠杆菌菌株。大肠杆菌 K12 W3110 将用作宿主菌株。对 LPS 生物合成途径至关重要并可能干扰 PglL O-糖基化的两个基因（wecA：初始糖基转移反应，waaL：新生多糖链与核心脂质 A 的连接）将被破坏。然后将证明在宿主菌株中异源表达 CPS14 的可行性。编码PglL和有利的CRM197变体的基因，以及CPS14异源表达所需的CPS14基因簇，将被引入突变宿主菌株中。将探索该工程菌株发酵过程中 CPS14-CRM197 缀合物的表达、纯化、表征和生物测定。 CRM197 缀合 CPS14 生产的成功示范将为生产其他肺炎链球菌血清型的 CPS 缀合物以及其他病原体的多糖奠定基础。鉴于 90 种肺炎链球菌血清型的 CPS 生物合成基因簇已被完全测序，多糖生物合成位点的可访问性将使我们的方法能够推广用于生产各种多糖结合疫苗。公共卫生相关性：细菌感染是全世界主要的健康问题之一。多糖形成包围细菌病原体的厚胶囊，是致病性的主要决定因素。随着对主要抗生素耐药性的不断出现，基于多糖的疫苗的开发为对抗传染病提供了一种有吸引力的方法。多糖最好与载体蛋白缀合作为缀合疫苗，以增强其功效。多糖结合疫苗生产的传统化学方法已经能够生产出目前临床使用的几种非常成功的结合疫苗。但传统方法生产步骤复杂、产量低、产品不纯，导致疫苗生产成本较高。本申请的目的是开发一种多糖结合疫苗生产的新方法。通过该方法，我们可以简便、高效、易于应用地获得多糖结合疫苗。该方法将首先以单一病原体为模型进行探索。然后所建立的方法可以很容易地应用于其他病原体。