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: 9127079
• 负责人: Peng George Wang
• 金额: $ 41.04万
• 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/9127079
• 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; 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.

描述(由申请人提供)：这项拟议的计划可被视为R01AI083754资助的竞争性续展，该资助名为“开发生产抗菌糖结合疫苗的新策略”，由ARRA于2009年7月30日至2011年7月29日资助。这项拟议的工作旨在解决疫苗开发中未得到满足的生物医学需求。抗菌疫苗的发展为防治细菌性疾病提供了一条诱人的途径。细菌表面定位多糖(PSS)具有作为疫苗预防细菌感染的巨大潜力。传统的多糖与载体蛋白化学结合制备PS-蛋白结合疫苗已成功地应用于临床，但仍存在组成不稳定、质量控制困难、效价不一致、生产成本高等问题。致命的问题是，这种方法不能产生结构定义良好的纯化学实体，在随后的结构-活性关系(SAR)研究中，这种实体可以与其免疫活性联系起来，而这在现代小分子药物发现计划中是常规的。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问题提供了新的途径。空肠弯曲菌的寡糖转移酶(PglB)是由Markus Aebi于2002年首次发现的，后来由GlycoVaxyn LLC开发，它能够将来自不同细菌的多种PS从其二磷酸-十一碳烯基转移到周质中一个共同的靶蛋白序列的ASN上。这种合成生物学方法与我们长期研究微生物多糖生物合成的努力很好地契合。因此，我们一直在ARRA R01AI083754下开发这种新方法。我们的努力导致了最近的一项重大技术突破，我们找到了一种简便的方法，将任何20-30kb的多糖生物合成基因簇克隆到表达载体中。现在，重组E.ColiK12菌株(与O-抗原基因簇、pglB和携带蛋白基因acrA分别整合在一个载体中)一次发酵就可以生产出结构明确的多糖-蛋白质生物结合物。例如，我们最近通过简单的大肠杆菌发酵生产了4.5 mg完全纯化的大肠杆菌O157O抗原多糖结合AcrA蛋白。这种生物结合物已经提供了很好的可能性，可以用来诱导奶牛或牛的抗体来杀死接种的O157大肠杆菌，因为这种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-蛋白质生物结合物和变异体，以及研究几种重要的生物结合物疫苗的SAR。目的1：PS蛋白生物结合物的生产：将两类细菌感染结合在一起。第一类包括O157、O104和宋内氏志贺氏菌；第二类包括金黄色葡萄球菌。目的：生产PS蛋白变异体我们的细菌蛋白N-糖基化平台允许我们改变多糖的长度、载体蛋白的长度、PS在载体蛋白上的密度以及任何免疫活性多肽或蛋白与载体蛋白的融合。这种PS蛋白变体将首次用于合成孔径雷达研究。目的3：PS-蛋白质生物结合物的免疫学研究与GSU的免疫学家同事合作(随着计划的展开，将有更多的国内和国际合作者)，将调查PS-蛋白质生物结合物的免疫学活性，目的是找到比目前传统方法更好的保护性疫苗。

项目成果

Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

• DOI: 10.1016/j.bmcl.2011.03.072
• 发表时间: 2011-09-01
• 期刊: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
• 影响因子: 2.7
• 作者: Li, Zijie;Cai, Li;Qi, Qingsheng;Styslinger, Thomas J.;Zhao, Guohui;Wang, Peng George
• 通讯作者: Wang, Peng George

Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.

• DOI: 10.1039/c5sc02025e
• 发表时间: 2015-10-01
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Li L;Liu Y;Ma C;Qu J;Calderon AD;Wu B;Wei N;Wang X;Guo Y;Xiao Z;Song J;Sugiarto G;Li Y;Yu H;Chen X;Wang PG
• 通讯作者: Wang PG

A Chemoenzymatic Histology Method for O-GlcNAc Detection.

用于 O-GlcNAc 检测的化学酶组织学方法。

• DOI: 10.1002/cbic.201700515
• 发表时间: 2017-12-14
• 期刊: Chembiochem : a European journal of chemical biology
• 作者: Aguilar AL;Hou X;Wen L;Wang PG;Wu P
• 通讯作者: Wu P

Comparing substrate specificity of two UDP-sugar pyrophosphorylases and efficient one-pot enzymatic synthesis of UDP-GlcA and UDP-GalA.

比较两个UDP糖焦磷酸酶的底物特异性以及UDP-GLCA和UDP-GALA的有效的一锅酶促合成。

• DOI: 10.1016/j.carres.2015.04.001
• 发表时间: 2015-06-26
• 期刊: CARBOHYDRATE RESEARCH
• 影响因子: 3.1
• 作者: Guo, Yuxi;Fang, Junqiang;Li, Tiehai;Li, Xu;Ma, Cheng;Wang, Xuan;Wang, Peng G.;Li, Lei
• 通讯作者: Li, Lei

Peptide adjacent to glycosylation sites impacts immunogenicity of glycoconjugate vaccine.

与糖基化位点相邻的肽会影响糖缀合物疫苗的免疫原性。

• DOI: 10.18632/oncotarget.19944
• 发表时间: 2018-01-02
• 期刊: Oncotarget
• 作者: Ma Z;Zhang H;Wang PG;Liu XW;Chen M
• 通讯作者: Chen M