Towards a New Generation of Glycoengineered Pneumococcal Bioconjugate Vaccines

项目摘要 肺炎球菌性肺炎仍然是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， 其由免疫原性载体蛋白共价连接到十三种肺炎球菌荚膜中的一种组成， 多糖尽管Prevnar-13显著降低了肺炎球菌疾病的负担，但它仅 预防90多种肺炎球菌血清型中的13种;此外，目前用于扩大 众所周知，血清型覆盖率很低，需要复杂合成化学来连接新的 肺炎球菌荚膜多糖与免疫原性载体蛋白结合。在过去的十年里， 一直在开创一种创新的方法来开发结合疫苗，大大简化了 糖缀合物的产生。这种糖工程策略，包括利用细菌 通过糖基化机制产生“生物缀合物”，消除了复杂的化学缀合的需要 方法通过使用缀合酶将多糖连接到大肠杆菌中的受体蛋白。 两种缀合酶PglB和PglL已经商业化用于产生生物缀合物，因为它们 能够将各种多糖转移到蛋白质中;然而，两者都不能转移 在还原端含有葡萄糖的多糖（生长多糖链的第一个糖）。 这一看似简单的观察结果具有巨大的意义，因为大约80%的肺炎球菌感染者， 胶囊在还原端含有葡萄糖。最近，我们已经确定并申请了第一个共轭 能够有效地将含有葡萄糖的肺炎球菌荚膜多糖转移到 还原端连接到受体蛋白。基于这一观察，我们将偶联我们的新型缀合酶， 技术与载体蛋白以前利用在共轭疫苗制剂，精简 产生具有更广泛血清型覆盖的上级肺炎球菌疫苗。重要的是我们的 糖工程策略不需要病原生物作为多糖的来源，也不需要化学物质 将多糖与蛋白质连接的反应。 本阶段I申请中的拟议研究将集中于（目标1）糖工程三 含有模块糖标签的商业载体蛋白（外毒素A、破伤风毒素片段C和CRM 197 用肺炎球菌荚膜多糖产生新的肺炎球菌生物缀合物疫苗 血清型8、9V、14和15 b。随后（目的2），我们将证明免疫原性和疗效 我们的肺炎球菌特异性生物结合疫苗与标准预防性治疗Prevnar-13相比。 我们的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， 开发大规模纯化方案，以获得我们的生物结合疫苗，以及临床前研究 为了进一步证明我们的下一代糖工程肺炎球菌的安全性、效力和有效性， 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.