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

描述（由申请人提供）：该项目可被视为R01AI083754项目的竞争性续期，标题为“开发一种生产抗菌糖结合疫苗的新策略”，该项目由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。细菌表面定位多糖（PSs）具有很大的潜力，可作为预防细菌感染的疫苗。虽然传统的多糖与载体蛋白化学偶联制备ps蛋白偶联疫苗已经产生了几种非常成功的用于临床的糖偶联疫苗，但它仍然存在批次组成不稳定、质量控制困难、效价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上定义良好的纯化学实体，在随后的结构-活性关系（SAR）研究中，可以将其与免疫活性联系起来，因为它在现代小分子药物发现项目中是常规的。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难题提供了新的途径。来自空肠弯曲杆菌（Campylobacter jejuni）的寡糖转移酶（Oligosaccharyltransferase, PglB），最早由Markus Aebi于2002年发现，后来由GlycoVaxyn LLC开发，被发现能够将多种PS（来自不同细菌）从二磷酸-去戊烯基形式转移到外质中目标蛋白的一致序列的Asn上。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相适应。因此，我们一直在ARRA R01AI083754下开发这种新方法。我们的努力取得了巨大的技术突破，我们研究出了一种简单的方法，可以将任何20至30 kb的多糖生物合成基因簇克隆到表达载体上。现在，重组大肠杆菌K12菌株（加入o抗原基因簇、pglB和携带蛋白基因acrA，各在一个载体中）通过一次发酵可以生产结构明确的多糖-蛋白生物偶联物。例如，我们最近从简单的大肠杆菌发酵中生产了4.5 mg完全纯化的大肠杆菌O157 o抗原多糖结合AcrA蛋白。这种生物偶联物已经提供了很好的可能性，可以用于诱导奶牛或牛体内的抗体，以杀死接种的大肠杆菌O157，因为疫苗可以经济地大规模生产。因此，本项目旨在生产一系列ps蛋白的生物偶联物和变异体，并研究几种重要的生物偶联疫苗的SAR。目标1:ps蛋白生物偶联物的生产：两类细菌感染将被附加。第一种包括大肠杆菌O157、O104和索尼氏志贺氏菌；第二种包括金黄色葡萄球菌。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。这些ps蛋白变体将首次用于SAR研究。与GSU的免疫学家同事合作（随着项目的展开，将有更多的国家和国际合作者），ps蛋白生物偶联物的免疫学活性将被研究，目标是找到比目前传统方法更好的保护性疫苗。

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.