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一种产生保护性免疫反应的有效手段，可以预防多种疾病。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。本申请的目的是探索最近建立的细菌蛋白o糖基化体系，以一种简便、有效和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一种高度混杂的蛋白pgl，它催化多糖从二磷酸脂供体转移到靶蛋白。概念验证实验为进一步探索这种结合疫苗开发系统提供了令人信服的证据。此外，该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理解，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。该STTR应用的第一阶段将以肺炎球菌病原体（肺炎链球菌血清型14）为模型证明该方法的可行性。我们计划将工作重点放在以下两个具体目标上：我们将建立一个基于脂质焦磷酸连接单糖的化学合成和相应CPS重复单元的酶组装的体外反应体系。除了确认pll对该特异性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，以获得最有利的pll底物序列。最后，多糖结合疫苗常用的载体蛋白（CRM197）将被设计为体内生产系统的受体底物。2. 我们的目标是获得一株产生CRM197结合CPS14的大肠杆菌菌株。大肠杆菌K12 W3110将被用作宿主菌株。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）对LPS生物合成途径至关重要，可能干扰PglL o糖基化的基因将被破坏。CPS14在宿主菌株中异种表达的可行性将得到证实。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偶联物的表达、纯化、鉴定和生物测定将进行探讨。CRM197偶联CPS14的成功生产将为从其他肺炎链球菌血清型中生产CPS偶联物以及从其他病原体中生产多糖奠定基础。鉴于90种肺炎链球菌血清型的CPS生物合成基因簇已经完全测序，多糖生物合成位点的可及性将使我们的方法推广到各种多糖结合疫苗的生产中。公共卫生相关性：细菌感染是世界范围内的主要健康问题之一。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。随着对主要抗生素耐药性的日益增加，开发基于多糖的疫苗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结合作为结合疫苗，以提高其效力。多糖结合疫苗的传统化学生产方法使几种目前临床使用的结合疫苗的生产非常成功。然而，传统方法的缺点是生产步骤复杂，产量低，产品不纯，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。该方法简便、高效、易于应用，可获得多糖结合疫苗。该方法将首先以单一病原体为模型进行探索。建立的方法可以很容易地应用于其他病原体。