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

描述(由申请人提供)：事实证明，多糖结合疫苗是产生保护性免疫反应的有效手段，从而预防多种疾病。传统的化学生产方法已经使临床上生产出了几种非常成功的结合疫苗。然而，传统的方法存在发酵多、纯化步骤多、产率低、化学偶联不特异等缺点，导致疫苗生产成本较高。本应用的目的是探索一种新近建立的细菌蛋白O-糖基化体系，以简便、高效和易于应用的方式获得多糖结合疫苗。这个细菌糖基化系统包括一种高度混杂的蛋白质PglL，它催化多糖类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这种系统提供了令人信服的证据。此外，这种方法的成功还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、化学和生化方法进行研究。这一STTR应用的第一阶段将使用肺炎链球菌病原体(14型肺炎链球菌)作为模型来证明这种方法的可行性。我们计划将工作集中在以下两个具体目标上：1.建立基于化学合成脂质焦磷酸连接单糖和相应CPS重复单位的酶组装的体外反应系统。除了确认PglL对这一特定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多肽偏好，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)将被设计成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。将以大肠杆菌K12 W3110作为宿主菌。两个基因(WECA：初始糖基转移反应，WAAL：新生的多糖链连接到核心-脂蛋白A)对内毒素的生物合成途径至关重要，可能干扰PglL O-糖基化。随后将论证CPS14在宿主菌中异源表达的可行性。编码PglL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。对该工程菌发酵产生的CPS14-CRM197结合物的表达、纯化、鉴定和生物检测进行了探索。CRM197结合物CPS14的成功生产将为从其他肺炎链球菌血清型和其他病原体生产CPS结合物奠定基础。鉴于90个肺炎链球菌血清型的CPS生物合成基因簇已经完全测序，多糖生物合成基因座的可获得性将使我们的方法在生产各种多糖结合疫苗方面得到推广。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。多糖在细菌病原体周围形成一层厚厚的包膜，是致病性的主要决定因素。随着对主要抗生素耐药性的增加，以多糖为基础的疫苗的开发为防治传染病提供了一条诱人的途径。多糖作为结合疫苗较好地结合到载体蛋白上，以提高其免疫效果。传统的化学方法生产多糖结合疫苗使目前临床上使用的几种非常成功的结合疫苗的生产成为可能。然而，传统方法生产步骤复杂、产率低、产品不纯，导致疫苗生产成本较高。本应用的目的是开发一种生产多糖结合疫苗的新方法。通过这种方法，我们可以方便、高效、易用地获得多糖结合疫苗。这一方法将首先以单一病原体为模型进行探索。因此，建立的方法可以很容易地应用于其他病原体。

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.