Development and testing of novel recombinant pnemococcal glyconjugate vaccines

肺炎链球菌，或肺炎球菌，可导致肺炎、败血症、脑膜炎等危及生命的疾病，并经常导致儿童耳部感染，导致听力丧失。肺炎链球菌在世界范围内造成了严重的发病率和死亡率，据保守估计，肺炎链球菌感染每年导致100多万儿童死亡。人们迫切需要一种廉价、广泛、长效的肺炎球菌疫苗。一种成功疫苗的一个决定性特征是能够以最小的副作用激发持久的保护性免疫。最成功的人类疫苗通常是糖结合的，因为蛋白质与糖结合在一起，既能诱导T细胞依赖的免疫反应，又能诱导独立的免疫反应，从而产生保护性和持久的免疫。目前获得许可的人糖结合疫苗的例子包括针对流感嗜血杆菌、脑膜炎奈瑟菌和一些肺炎链球菌的疫苗，其中多糖与免疫原载体蛋白化学偶联。通过化学偶联设计的传统糖结合疫苗要求从病原体中分离出糖，通过剥离表面成分来解毒，并且仍然存在足够的量以与蛋白质化学偶联。这些程序涉及严苛的化学处理，既耗时又昂贵。此外，每一步产生的物质需要验证纯度，糖结合疫苗批次之间的差异是常见的。目前获得许可的肺炎球菌糖结合疫苗是有问题的，因为它们只覆盖所有肺炎链球菌菌株的一小部分。尽管有基于保守蛋白质的候选疫苗，但这些疫苗往往不能产生长期保护，特别是对主要目标人群儿童进行免疫所需的长期保护。理想情况下，应该生产一种基于保守的肺炎球菌蛋白偶联到荚膜多糖多糖上的糖结合疫苗，但到目前为止，这已被证明是技术上的挑战。最近，我们(和合作者)开发了一种新的构建糖结合疫苗的方法，包括克隆广泛使用的工作微生物大肠杆菌中的所有成分。重组过程被称为蛋白质葡聚糖偶联技术(PGCT)，涉及在大肠杆菌中处理候选蛋白质和葡聚糖在质粒载体中与偶联酶一起生产取之不尽的疫苗。PGCT可以通过一步纯化程序生产纯化疫苗，这降低了成本，而且由于蛋白质和多糖的多种组合可以偶联在一起，因此可以在疫苗范围内产生和测试更大的灵活性。我们将使用PGCT来生产和测试六种优秀的候选蛋白，这些候选蛋白与不同组合的肺炎球菌荚膜多糖偶联。这些疫苗将在小鼠肺炎球菌感染模型中进行测试，以确定它们对其他致命剂量的相对保护作用。此外，还将在小鼠模型中测试疫苗对肺炎球菌携带的影响。这项研究中产生的新疫苗还将与现有肺炎球菌疫苗(如Prevnar13)的效力进行比较。将对试验之间的数据进行评估，以得出PGCT生产的最有效的糖结合疫苗组合。此外，本研究中PGCT的发展将为进一步构建肺炎链球菌糖结合疫苗和针对其他重要感染源的疫苗提供专业知识和知识基础。

Streptococcus pneumoniae, or the pneumococcus, can cause life-threatening diseases such as pneumonia, septicaemia, meningitis and frequently causes ear infections in children which can lead to hearing loss. S. pneumoniae is responsible for significant morbidity and mortality worldwide and by conservative estimates pneumococcal infections cause over one million deaths of children annually. An inexpensive, broad-range, long-lasting pneumococcal vaccine is desperately required.A defining characteristic of a successful vaccine is the ability to evoke long-lasting protective immunity with minimal side effects. The most successful human vaccines are often glycoconjugate as the combination of a protein coupled to a sugar glycan induces both a T-cell dependent and independent immune response evoking a protective and lasting immunity. Examples of currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisserria meningitidis and some Streptococcus pneumoniae strains, in which glycans are chemically coupled to immunogenic carrier proteins.Traditional glycoconjugate vaccine design by chemical conjugation requires that the glycan from the pathogenic organism be isolated, detoxified by stripping out surface components, and still be present in sufficient amounts to be chemically coupled to a protein. The procedures involve harsh chemical treatments, are time consuming and expensive. In addition, the material generated at each step needs to be verified for purity, and variation between batches of glycoconjugate vaccine is common. Current licensed pneumococcus glycoconjugate vaccines are problematic as they only cover a fraction of all S. pneumoniae strains. Although there are vaccine candidates based on conserved proteins, these vaccines often do not produce long-term protection that is especially required to immunize the main target population, children. Ideally, a glycoconjugate vaccine based on conserved pneumococcal proteins coupled to the capsular polysaccharide glycan should be produced, but to date this has proved technically challenging.Recently, we (and collaborators) have developed a new approach for constructing glycoconjugate vaccines involving cloning all components in the widely used "work-horse" microbe E. coli. The recombinant process is termed Protein Glycan Coupling Technology (PGCT) and involves processing the candidate protein and glycan in plasmid vectors in E. coli along with a coupling enzyme to produce an inexhaustible supply of vaccine. PGCT can produce purified vaccine in a one-step purification procedure, which reduces costs, and because multiple combinations of protein and glycans can be coupled together, a greater flexibility in the range of vaccines can be generated and tested. We will use PGCT to produce and test six outstanding protein candidates coupled to different combinations of pneumococcal capsular polysaccharide. These vaccines will be tested in the murine pneumococcal infection model for their relative protection against an otherwise lethal dose. Additionally, the vaccines will be tested for their effect on the carriage of pneumococci in the murine model. The new vaccines generated in this study will also be compared to the efficacy of existing pneumococcal vaccines such as Prevnar13. Data between experiments will be evaluated to derive the most efficacious glycoconjugate vaccine combination produced by PGCT. Additionally, the development of PGCT in this study will provide the expertise and knowledge base to make the technology more widely applicable to construct further S. pneumoniae glycoconjugate vaccines and vaccines against other important infectious agents.