Towards a New Generation of Glycoengineered Pneumococcal Bioconjugate Vaccines

项目总结 肺炎球菌肺炎仍然是两名5岁以下儿童细菌性肺炎的主要原因 年龄和65岁以上的成年人。标准的预防性治疗是结合疫苗Prevnar-13， 它由免疫原载体蛋白组成，共价连接到13个肺炎球菌囊膜中的一个上 多糖类。尽管Prevnar-13大大减轻了肺炎球菌疾病的负担，但它只 对90多种肺炎球菌血清型中的13种有保护作用；此外，目前用于扩大 众所周知，血清型覆盖速度很慢，需要复杂的合成化学方法来连接新的 肺炎链球菌衣壳多糖为免疫原性载体蛋白。在过去的十年里，我们有 我开创了一种结合疫苗开发的创新方法，大大简化了 生产糖共轭化合物。这一糖工程战略，包括利用细菌 糖基化机制可产生“生物偶联物”，无需复杂的化学偶联反应。 方法在大肠杆菌中利用偶联酶将多糖连接到受体蛋白上。 两种结合酶PglB和PglL已被商业化利用来产生生物结合物，因为它们 都能将各种各样的多糖转移到蛋白质上；然而，两者都不能转移 还原末端含有葡萄糖的多糖(生长中的多糖链中的第一个糖)。 这一看似简单的观察具有巨大的意义，因为大约80%的肺炎球菌 胶囊的还原端含有葡萄糖。最近，我们已经确定并申请了第一个共轭 一种能够有效地将含有葡萄糖的肺炎链球菌壳多糖转移到 还原末端为受体蛋白。在此基础上，我们将偶联我们的新型偶联酶 以前在结合疫苗配方中使用载体蛋白的技术，简化了 产生一种具有更广泛的血清型覆盖范围的优质肺炎球菌疫苗。重要的是，我们的 糖工程策略不需要病原体作为多糖或化学物质的来源 将多糖与蛋白质联系起来的反应。 在这一阶段的应用中拟议的研究将集中在(目标1)糖工程三个方面。 商业载体蛋白(外毒素A、破伤风毒素片段C和CRM197)含有模块化糖标签 肺炎链球菌壳多糖制备新型肺炎链球菌生物结合疫苗 血清型8、9V、14和15b型。随后(目标2)我们将展示其免疫原性和有效性。 我们的肺炎球菌特异性生物结合疫苗与标准预防疗法Prevnar-13进行了比较。 我们第二阶段资金的下一步是扩大我们的生物结合疫苗中包括的血清型覆盖范围， 制定大规模纯化方案以获得我们的生物结合疫苗，以及临床前研究 进一步证明我们的下一代糖工程肺炎球菌的安全性、有效性和有效性 生物结合疫苗。

PROJECT SUMMARY Pneumococcal pneumonia remains the leading cause of bacterial pneumonia in both children under 5 years of age and adults over 65 years of age. The standard, preventative therapy is the conjugate vaccine, Prevnar-13, which consists of an immunogenic carrier protein covalently attached to one of thirteen pneumococcal capsular polysaccharides. Although Prevnar-13 has significantly reduced the burden of pneumococcal disease, it only protects against 13 of the 90 plus pneumococcal serotypes; furthermore, current methods employed to expand the serotype coverage are notoriously slow requiring complex synthetic chemistries to link a new pneumococcal capsular polysaccharide to the immunogenic carrier protein. Over the last decade, we have been pioneering an innovative approach to conjugate vaccine development that drastically simplifies the production of glycoconjugates. This glycoengineering strategy, consisting of the exploitation of bacterial glycosylation machineries to generate “bioconjugates”, eliminates the need of intricate chemical conjugation methods by employing conjugating enzymes to attach polysaccharides to acceptor proteins in Escherichia coli. Two conjugating enzymes, PglB and PglL, have been commercially utilized to generate bioconjugates as they are able to transfer a wide variety of polysaccharides to proteins; however, neither are able to transfer polysaccharides containing glucose at the reducing end (the first sugar of a growing polysaccharide chain). This seemingly simple observation has enormous implications as approximately 80% of pneumococcal capsules contain glucose at the reducing end. Recently, we have identified and patented the first conjugating enzyme that is able to efficiently transfer pneumococcal capsular polysaccharides containing glucose at the reducing end to an acceptor protein. Based on this observation, we will couple our novel conjugating enzyme technology with carrier proteins previously utilized in conjugate vaccine formulations, streamlining the generation of a superior pneumococcal vaccine with broader serotype coverage. Importantly, our glycoengineering strategy does not require pathogenic organisms as a source of polysaccharide nor chemical reactions to link polysaccharides to proteins. The proposed research in this phase I application will focus on (Aim 1) glycoengineering three commercial carrier proteins (exotoxin A, tetanus toxin fragment C, and CRM197) to contain a modular glycotag with pneumococcal capsular polysaccharides generating a new bioconjugate vaccine for pneumococcal serotypes 8, 9V, 14, and 15b. Subsequently (Aim 2) we will demonstrate the immunogenicity and efficacy of our pneumococcal specific bioconjugate vaccine compared to the standard preventative therapy Prevnar-13. Our next step for phase II funding is to expand the serotype coverage included in our bioconjugate vaccine, develop a large-scale purification scheme for obtaining our bioconjugate vaccine, as well as pre-clinical studies to further demonstrate the safety, potency, and efficacy of our next generation glycoengineered pneumococcal bioconjugate vaccine.