Development and application of an Advanced Glycan Production Platform

疫苗是人类在对抗感染方面取得的最大成功之一。它们有助于减少甚至根除天花和小儿麻痹症等严重传染病。疫苗还能有效地保护农场和伴侣动物免受可能伤害或杀死它们的疾病的侵害（给动物接种疫苗也能保护人类免受感染）。健康饲养的牲畜对经济和社会繁荣至关重要。除了降低患者和动物的死亡率外，疫苗的使用还减少了抗生素的使用，从而减少了抗菌素耐药性的传播。然而，我们缺乏许多传染病的疫苗，我们迫切需要针对新出现的病毒和多重耐药细菌的疫苗。大多数现代疫苗的工作原理是将病毒或细菌的一个小成分（“亚基”）注射到病人体内。这会刺激他们的免疫系统来抵御疾病。这些成分可以是糖或蛋白质。目前很难以可控的方式做到这一点。特别是，细菌的多糖糖衣（聚糖）（它可以制作出色的疫苗）的制备和与关键蛋白质靶点的偶联非常具有挑战性。我们的项目将通过开发一种新技术，在简单、安全的大肠杆菌细胞中高效、忠实地生产聚糖和基于聚糖的疫苗，从而克服这一问题，大肠杆菌细胞充当迷你疫苗工厂。在我们的项目中，我们使用新的合成生物学方法来改善聚糖和基于聚糖的产品的生产，如用于诊断和疫苗。这将涉及设计、构建和优化DNA序列的现代方法，以及测试许多不同的细胞调节修改以发现和利用最有用的DNA组合的新方法。这些将结合在一起，以优化定制大肠杆菌宿主菌株的聚糖生产。作为原理证明，我们将通过开发针对猪链球菌和布鲁氏菌两种主要动物病原体（也感染人类）的示范疫苗来测试我们的新聚糖生产平台。猪链球菌引起严重的全身性疾病，表现为与脑膜炎和肺炎相关的迅速致命的败血症，是养猪业的一个全球性问题。布鲁氏菌病是一种影响牛、绵羊、山羊和猪的高度传染性疾病。这些都是养殖动物的主要疾病，如果传播给人类可能是致命的。养活世界上不断增长的人口所需要的现代集约化农业技术，增加了牲畜之间可传染给人类的传染病的风险。显然需要低成本的猪链球菌和布鲁氏菌聚糖疫苗。本研究将利用新聚糖表达技术优化表达的猪链球菌荚膜多糖和布鲁氏菌属脂多糖制备靶向疫苗。此外，我们已确定与适当的合作伙伴进行临床前试验，以促进疫苗的生产和评估，以及本研究的努力。在这个项目结束时，我们将使用新的合成生物学方法来开发一个技术平台，在大肠杆菌中生产细菌聚糖，这将促进新一代诊断和疫苗的生产。

Vaccines are one of humanity's greatest successes in combating infections. They have helped to reduce or even eradicate severe infectious diseases such as smallpox and polio. Vaccines are also effective in protecting farm and companion animals from diseases that would otherwise harm or kill them (vaccinating animals protects humans from infection too). Healthily maintained livestock are essential for economic and societal prosperity. In addition to reducing mortality for patients and animals, vaccine use reduces antibiotic usage diminishing the spread of antimicrobial resistance. However, we lack vaccines for many infectious diseases, and we urgently need vaccines against newly emerging viruses and multi-drug resistant bacteria.Most modern vaccines work by taking one small component of a virus or bacterium (a "subunit") and injecting this into the patient. This stimulates their immune system to protect against the disease. These components can be sugars or proteins. It is currently hard to do this in a controlled way. In particular, the polysaccharide sugar coats (glycans) of bacteria (which make excellent vaccines) are very challenging to prepare and to couple to key protein targets.Our project will overcome this by developing a new technology to produce glycans and glycan-based vaccines efficiently and faithfully in simple, safe types of E. coli cells that act as mini vaccine factories. In our project, we use new synthetic biology approaches to improve the production of glycans and glycan-based products such as for diagnostics and vaccines. These will involve modern approaches to design, construct and optimise DNA sequences and a new way to test many different modifications of cell regulation to find and exploit the most useful DNA combinations. These will be combined to optimise glycan production in bespoke E. coli host strains.As proof of principle, we will test our new glycan production platform by developing exemplar vaccines against two major pathogens of animals that also infect humans, Streptococcus suis and Brucella species. S. suis causes severe systemic disease manifested as a rapidly fatal sepsis associated with meningitis and pneumonia and is a global problem for the pig industry. Brucellosis is a highly contagious disease that affects cattle, sheep, goats and pigs. These are major diseases of farmed animals and can be fatal if transmitted to humans. Modern intensive farming techniques, needed to feed the world's growing population, increase the risk of infectious disease among livestock that can be transmitted to humans. There is a clear need for low-cost S. suis and Brucella glycan-based vaccines. Here, we will use the optimally expressed S. suis capsular polysaccharides and Brucella species lipopolysaccharide from the new glycan expression technology to produce targeted vaccines. In addition, we have an identified pipeline into pre-clinical trials with appropriate partners to facilitate the vaccine production and evaluation, and the endeavours from this study.At the conclusion of this project, we will have used novel synthetic biology approaches to develop a technology platform to produce bacterial glycans in E. coli which will facilitate the production of a new generation of diagnostics and vaccines.