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: 8439987
• 负责人: Peng George Wang
• 金额: $ 36.9万
• 依托单位: GEORGIA 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439987
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotics; Antibodies; Bacteria; Bacterial Infections; Bacterial Proteins; Biogenesis; Campylobacter jejuni; Carrier Proteins; Cattle; Cells; Chemicals; Clinical; Communicable Diseases; Complex; Conjugate Vaccines; Consensus Sequence; Development; Epitopes; Escherichia coli; Escherichia coli K12; Escherichia coli O157; Fermentation; Funding; Gene Cluster; Gene Proteins; Genes; Glycoconjugates; Glycoproteins; Goals; Grant; Health; Immunologist; Infection; International; Intestines; Investigation; Length; Link; Methods; Modeling; O Antigens; Peptides; Polysaccharides; Process; Production; Proline; Proteins; Quality Control; Recombinants; Resistance; Series; Shigella sonnei; Staphylococcus aureus; Surface; System; Techniques; Thick; Time; Vaccine Production; Vaccines; Variant; Virulence Factors; Work; base; capsule; cost; density; dolichyl-diphosphooligosaccharide - protein glycotransferase; drug discovery; expression vector; fighting; glycosylation; killings; link protein; microbial; novel; novel strategies; pathogen; periplasm; prevent; programs; research and development; small molecule; synthetic biology; vaccine development; vector

DESCRIPTION (provided by applicant): This proposed program can be considered as a competitive renewal of grant R01AI083754, titled "Development of A Novel Strategy to Produce Antibacterial Glycoconjugate Vaccines", funded under ARRA from 7/30/2009 - 7/29/2011. The proposed work is aiming at solving an unmet biomedical need in the development of vaccines. Development of antibacterial vaccines provides an attractive approach for fighting bacterial diseases. Surface-located Polysaccharides (PSs) of bacteria have great potentials to be used as vaccines for preventing bacterial infections. Although traditional chemical conjugation of polysaccharides with carrier proteins to make PS-protein conjugate vaccines has resulted in several highly successful glycoconjugate vaccines for the clinical use, it still suffers variable batch-to-batch composition, difficult quality control, inconsistent potency and high production cost. The fatal problem is that such approach does not produce a structurally well defined, pure chemical entity, which can be linked to its immunological activity in subsequent structural- activity relationship (SAR) investigation, as it is routinely done in modern drug discovery programs for small molecules. The discovery and further development of bacterial protein N- glycosylation system have provided a novel approach to solve this biomedical problem. Oligosaccharyltransferase (PglB) from Campylobacter jejuni, which was first discovered by Markus Aebi in 2002, later developed by GlycoVaxyn LLC, was found to be able to transfer a variety of PS (from different bacteria) from its diphospho-undecaprenyl forms to the Asn of a consensus sequence of the target protein in the periplasm. Such synthetic biology approach fits well with our long-term efforts on studying the biosynthesis of microbial polysaccharides. Thus we have been developing this novel approach under ARRA R01AI083754. Our efforts resulted in a recent huge technique breakthrough after we worked out a facile method to clone any 20 to 30 kb polysaccharide biosynthesis gene clusters into an expression vector. Now structurally well-defined polysaccharide-protein bioconjugate can be produced by one-shot fermentation of recombinant E. coli K12 strain (incorporated with O-antigen gene cluster, pglB and carry protein gene acrA, each in one vector). For example, we recently produced 4.5 mg fully purified E. coli O157 O-antigen polysaccharide conjugated AcrA protein from simple E. coli fermentation. Such a bioconjugate already offers good possibility to be used to induce antibodies in cows or cattle to kill an inoculum of E. coli O157, since the vaccines can be produced in large scale economically. Therefore, the program aims to produce a series of PS-protein bioconjugates and variants, as well as study the SAR of several important bioconjugate vaccines. Aim 1: Production of PS-protein bioconjugates: Two classes of bacterial infections will be attached. The first includes E. coli O157, O104 and Shigella sonnei; the second includes Staphylococcus aureus. Aim 2: Production of PS-protein variants Our bacterial protein N-glycosylation platform allow us to change the length of polysaccharides, the length of the carrier proteins, the density of PS on the carrier protein and fusion of any immunologically active peptide or protein to the carrier protein. Such PS-protein variants will be available for the first time for SAR investigatio. Aim 3: Immunological studies on PS-protein bioconjugates Collaborating with immunologist colleagues at GSU (many more national and international collaborators as the program unfolds), the immunological activities of PS-protein bioconjugates will be investigated with the goal of finding better protective vaccines than current conventional approaches can offer. PUBLIC HEALTH RELEVANCE: Bacterial infections are one of the major health problems worldwide. With the increasing emergence of resistance towards major antibiotics, development of polysaccharide based vaccines provides an attractive approach for fighting the infectious diseases. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, have been used as vaccines for preventing bacterial infections, and they were always linked to proteins to enhance their efficacy. The traditional chemical approach for producing such polysaccharide-protein linked vaccines suffers from complex production steps, low yields and impure products, thus leading to high costs of vaccines. The objective of this application is to develop a novel method for polysaccharide-protein linked vaccine production, by one-shot bacteria fermentation. 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）：该拟议计划可视为授予R 01 AI 083754的竞争性更新，标题为“开发生产抗菌糖缀合物疫苗的新策略”，由ARRA于2009年7月30日至2011年7月29日资助。拟议的工作旨在解决疫苗开发中未满足的生物医学需求。 抗菌疫苗的开发为对抗细菌性疾病提供了一种有吸引力的方法。细菌表面多糖作为疫苗预防细菌感染具有很大的潜力。尽管传统的多糖与载体蛋白的化学缀合以制备PS-蛋白缀合物疫苗已经导致几种非常成功的用于临床应用的糖缀合物疫苗，但是其仍然遭受批次间组成可变、质量控制困难、效力不一致和生产成本高的问题。致命的问题是，这种方法不能产生结构明确的纯化学实体，其可以在随后的结构-活性关系（SAR）研究中与其免疫活性相关联，如在现代小分子药物发现计划中常规进行的那样。细菌蛋白N-糖基化系统的发现和进一步发展为解决这一生物医学问题提供了新的途径。 由Markus Aebi于2002年首次发现的、后来由GlycoVaxyn LLC开发的来自空肠弯曲杆菌的寡糖基转移酶（PglB）被发现能够将多种PS（来自不同细菌）从其二磷酸-十一异戊二烯形式转移到周质中靶蛋白的共有序列的Asn。这种合成生物学方法与我们长期致力于微生物多糖生物合成的研究非常吻合。因此，我们一直在ARRA R 01 AI 083754下开发这种新方法。我们的努力导致了最近的一个巨大的技术突破后，我们制定了一个简单的方法，任何20至30 kb的多糖生物合成基因簇克隆到表达载体。目前，利用重组大肠杆菌一次发酵法可以生产出结构明确的多糖-蛋白生物结合物。coli K12菌株（在一个载体中整合有O-抗原基因簇、pglB和载体蛋白基因acrA）。例如，我们最近生产了4.5 mg完全纯化的E。coliO 157 O-抗原多糖结合AcrA蛋白。大肠杆菌发酵这种生物缀合物已经提供了用于在母牛或牛中诱导抗体以杀死E. coli O 157，因为疫苗可以经济地大规模生产。 因此，该计划旨在生产一系列PS-蛋白生物缀合物和变体，以及研究几种重要生物缀合物疫苗的SAR。目的1：生产PS-蛋白生物缀合物：将附上两类细菌感染。第一种包括E.大肠杆菌O 157、O 104和宋内志贺菌;其次为金黄色葡萄球菌。目标二：我们的细菌蛋白N-糖基化平台允许我们改变多糖的长度、载体蛋白的长度、载体蛋白上PS的密度以及任何免疫活性肽或蛋白与载体蛋白的融合。这种PS蛋白变体将首次用于SAR研究。目标三：与GSU的免疫学家同事合作（随着计划的展开，更多的国家和国际合作者），PS蛋白生物缀合物的免疫活性将被研究，目的是找到比目前传统方法更好的保护性疫苗。 公共卫生相关性：细菌感染是全球主要的健康问题之一。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有吸引力的方法。多糖，形成一个厚厚的胶囊，包围细菌病原体，已被用作预防细菌感染的疫苗，他们总是连接到蛋白质，以提高其效力。用于生产这种多糖-蛋白质连接的疫苗的传统化学方法具有生产步骤复杂、产率低和产品不纯的缺点，从而导致疫苗的高成本。本申请的目的是开发一种通过一次性细菌发酵生产多糖-蛋白质连接疫苗的新方法。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。该方法将首先用模式病原体进行探索。所建立的方法也可方便地应用于其他病原体的检测。