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）：该提出的计划可以被视为授予R01AI083754的竞争性更新，标题为“制定了生产抗菌糖壳偶然疫苗的新型策略”，从7/30/2009-2009-7/29/2011资助了ARRA。拟议的工作旨在解决疫苗开发中未满足的生物医学需求。 抗菌疫苗的开发为抗击细菌疾病提供了有吸引力的方法。细菌的表面划分的多糖（PS）具有巨大的潜力，可用作预防细菌感染的疫苗。尽管传统的化学化学偶联性与载体蛋白与载蛋白结合疫苗的持续化导致几种非常成功的糖缀合疫苗用于临床用途，但它仍然会遭受可变的批次到批量组成，难以控制的质量控制，不一致的效力和高生产成本。致命的问题是，这种方法不会产生结构良好的纯化学实体，这可以与其在随后的结构活动关系（SAR）研究中与其免疫学活性联系起来，因为它在小分子的现代药物发现计划中通常会进行。细菌蛋白N-糖基化系统的发现和进一步发展提供了一种解决这一生物医学问题的新方法。 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.这种合成生物学方法非常符合我们研究微生物多糖生物合成的长期努力。因此，我们一直在根据ARRA R01AI083754开发这种新颖的方法。我们的努力在我们制定了一种轻松的方法来克隆任何20至30 kb多糖生物合成基因簇中的任何一个巨大的技术突破中，这导致了巨大的技术突破。现在，可以通过一试重组大肠杆菌K12菌株（与O-抗原基因簇，PGLB和携带蛋白基因ACRA掺入重组大肠杆菌K12菌株（掺入），可以产生结构明确定义的多糖蛋白质生物缀合物。例如，我们最近产生了4.5 mg完全纯化的大肠杆菌O157 O-抗原多糖从简单的大肠杆菌发酵中共轭ACRA蛋白。这样的生物轭已经提供了很好的可能性，可以用来诱导牛或牛杀死大肠杆菌O157的接种物，因为该疫苗可以在经济上大规模生产。 因此，该计划旨在生产一系列PS蛋白生物偶联物和变体，并研究几种重要的生物偶联疫苗的SAR。目标1：PS蛋白生物缀合物的产生：将附着两类细菌感染。第一个包括大肠杆菌O157，O104和Shigella Sonnei；第二个包括金黄色葡萄球菌。目标2：生产PS蛋白质变体我们的细菌蛋白N-糖基化平台使我们能够改变多糖的长度，载体蛋白的长度，载体蛋白上PS的密度以及任何免疫活性活性肽或蛋白质的任何免疫活性活性肽或蛋白质的融合。这种PS蛋白质变体将首次进行SAR调查。 AIM 3：关于PS蛋白生物共轭物的免疫学研究，与GSU的免疫学家同事（随着该计划的展开）合作，PS蛋白生物缀合物的免疫学活动将与当前的传统方法相比，将对更好的保护疫苗进行研究。 公共卫生相关性：细菌感染是全球主要的健康问题之一。随着对主要抗生素的耐药性的越来越多，基于多糖的疫苗的开发为抵抗传染病的有吸引力的方法。多糖形成围绕细菌病原体的厚胶囊，已被用作预防细菌感染的疫苗，并且它们始终与蛋白质相关，以增强其功效。这种产生这种多糖蛋白接种疫苗的传统化学方法遭受了复杂的生产步骤，低收益和不纯净的产品，因此导致了高昂的疫苗成本。该应用的目的是通过一次性细菌发酵开发一种新型的多糖蛋白蛋白连接疫苗的方法。通过这种方法，我们可以以便捷，高效且易于适用的方式获得多糖偶联疫苗。首先将使用模型病原体探索此方法。然后，已建立的方法可以轻松地应用于其他病原体。