Universal protection against Streptococcus pneumoniae by recombinant glycoconjugate vaccines

疫苗是预防传染病的关键组成部分，消除了人类面临的一些最危险的疾病。在低收入国家，这种情况越来越多，因为疫苗可以改变健康儿童的可能性。肺炎链球菌可导致肺炎、败血症和脑膜炎等危及生命的疾病。肺炎链球菌在世界范围内造成了严重的发病率和死亡率，每年有100多万儿童死亡。耐药肺炎链球菌菌株的出现和迅速传播进一步强调了预防肺炎链球菌感染的必要性。廉价、大范围、长效肺炎球菌疫苗是当前全球的当务之急，对LMIC国家的影响最大，因为在这些国家，现有疫苗经常得不到充分利用，肺炎链球菌感染仍然是儿童死亡和发病的一个重要原因。一种成功疫苗的一个决定性特征是能够以最小的副作用唤起持久的保护性免疫。最成功的人类疫苗通常是糖偶联物，这是一种蛋白质与糖聚糖的组合，因为它们为免疫系统提供了多种触发因素，并延长了疫苗的寿命。目前人类糖结合疫苗的例子包括针对流感嗜血杆菌、脑膜炎奈瑟菌和肺炎链球菌菌株的疫苗。这些疫苗是通过化学方法制造的，这既耗时又昂贵。此外，目前的肺炎球菌糖结合疫苗只对所有肺炎链球菌株的一小部分具有保护作用。理想情况下，为了提高疫苗保护的所有肺炎链球菌的比例，针对肺炎链球菌的糖结合疫苗应该将糖成分与存在于所有菌株中的肺炎链球菌蛋白联系起来，但到目前为止，这被证明是具有技术挑战性的。最近，我们开发了一种构建糖结合疫苗的新方法，该方法涉及对大肠杆菌进行遗传改变，使其成为生产糖结合疫苗的细胞工厂。这项技术被称为蛋白质多糖偶联技术(PGCT)，涉及制造一种能够产生候选蛋白质和多糖的大肠杆菌菌株，以及一种将蛋白质和多糖偶联在一起的酶，以生产取之不尽、价格低廉的疫苗。PGCT可以通过一步纯化程序生产纯化疫苗，这降低了成本，而且由于可以生产多种蛋白质和多糖组合，因此可以在疫苗范围内产生和测试更大的灵活性。然而，到目前为止，我们还不知道使用PGCT生产的疫苗中使用的最佳肺炎链球菌蛋白能够诱导对肺炎链球菌感染的最高水平的保护。在这项研究中，我们将使用新技术系统地筛选所有肺炎链球菌蛋白，以确定由肺炎链球菌蛋白与肺炎链球菌多糖(胶囊多糖)偶联而成的“双击式”糖结合疫苗的最佳候选者。我们将从筛选的前50名候选者中挑选出可以用PGCT连接到肺炎链球菌胶囊多糖上的候选分子进行测试，以制备有效的重组糖结合物。然后，最有希望的疫苗将在肺炎链球菌感染的小鼠模型中进行测试，以找出哪些疫苗能够最好地预防感染。生产的新疫苗还将与Prevenar13等市场领先疫苗的疗效进行比较。这些实验将确定最适合包含在使用PGCT制造的新型肺炎链球菌疫苗中的蛋白质，或用于其他新的疫苗方法。此外，本研究中PGCT的发展将提供专业知识和知识基础，使该技术更广泛地适用于制造针对其他重要传染病病原体的糖结合疫苗。

Vaccines are a critical component of defence against infectious disease, and have eliminated some of the most dangerous diseases that have faced humanity. This is increasingly the case in low income countries, where vaccines can transform the likelihood of healthy childhoods. Streptococcus pneumoniae can cause life-threatening diseases such as pneumonia, septicaemia and meningitis. S. pneumoniae is responsible for significant morbidity and mortality worldwide with over one million deaths of children annually. The emergence and rapid spread of antibiotic-resistant S. pneumoniae strains has further emphasised the need for prevention of S. pneumoniae infections. An inexpensive, broad-range, long-lasting pneumococcal vaccine is a current global imperative, and be of most impact to LMIC countries as this is where existing vaccines are often under utilised and S. pneumoniae infections remain a significant cause of childhood mortality and morbidity. 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 glycoconjugates, which are combinations of a protein coupled to a sugar glycan, as these provide multiple triggers for the immune system, and increases the lifetime of the vaccine. Examples of current human glycoconjugate vaccines include vaccines against Haemophilus influenzae, Neisserria meningitidis and S. pneumoniae strains. These vaccines are made chemically which is time consuming and expensive. Furthermore, the current pneumococcus glycoconjugate vaccines only protect against a fraction of all S. pneumoniae strains. Ideally, to improve the proportion of all S. pneumoniae strains that the vaccine protects against, a glycoconjugate vaccine against S. pneumoniae should link the sugar component to S. pneumoniae proteins that are present in all strains, but to date this has proved technically challenging to achieve.Recently, we have developed a new approach for constructing glycoconjugate vaccines involving genetically altering the bacterium E. coli so that they act as cellular factories for the production of glycoconjugate vaccines. This is termed Protein Glycan Coupling Technology (PGCT), and involves making an E. coli strain that can produce the candidate protein and glycan, along with an enzyme that couples the protein and glycan together to produce an inexhaustible and inexpensive 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 produced, a greater flexibility in the range of vaccines can be generated and tested. However, as yet we do not know the best S. pneumoniae proteins to use in a vaccine made using PGCT that are able to induce the highest level of protection against S. pneumoniae infections. In this study we will use new technologies to systematically screen all S. pneumoniae proteins to identify the best candidates for a "double hit" glycoconjugate vaccine consisting of a S. pneumoniae protein coupled to S. pneumoniae glycan (capsular polysaccharide). We will select the top 50 candidates from the screen to test which can be linked using PGCT to S. pneumoniae capsule glycan to make effective recombinant glycoconjugates. The most promising vaccines will then be tested in mouse models of S. pneumoniae infection to find which ones are best able to prevent infections. The new vaccines generated will also be compared to the efficacy of market leading vaccines such as Prevenar13. These experiments will identify the most suitable proteins for inclusion in a novel S. pneumoniae vaccine made using PGCT, or for other novel vaccine approaches. Additionally, the development of PGCT in this study will provide the expertise and knowledge base to make the technology more widely applicable for making glycoconjugate vaccines against other important infectious agents.