Development and application of an Advanced Glycan Production Platform

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.

Bacterial glycans are key sugar-like structures frequently found on cell surfaces that come in infinite varieties. Because of their surface location they often have important roles in pathogenesis and have substantial diagnostic and vaccine applications. However, the cloning, production and exploitation of glycans has lagged behind protein and nucleic acid counterparts. This is because they invariably consist of multi-gene loci which encode several enzymes that synthesise complex glycan structures. Central to exploitation is the ability to clone and express glycans in host cells such as E. coli, the workhorse of biotechnology. Building on excellent preliminary studies expressing the Campylobacter jejuni N-linked pgl glycan, we will combine novel synthetic biology approaches to develop a new platform technology for optimal glycan expression using (i) 'refactoring' of glycan clusters (deconstructing and rebuilding in a tunable modular format) and combinatorial optimisation, (ii) engineered regulation, and (iii) our bank of rationally designed E. coli host strains. As a test bed for the platform technology we will clone and express key glycans from the major zoonotic animal pathogens Streptococcus suis (serotype 2 capsular polysaccharides) and Brucella species (lipopolysaccharide, LPS) where we have proven expertise. Currently no satisfactory vaccine exists for either pathogen. Once optimal glycan expression is established, we will use our proprietary Protein Glycan Coupling Technology or alternative membrane vesicle technology to produce much needed, low-cost S. suis and Brucella vaccines. The proposal will demonstrate the optimum expression of a broad range of glycans including capsular polysaccharides from a Gram-positive pathogen, LPS and an N-linked glycosylation system from Gram-negative pathogens. The principles and technology developed in this study will be more widely applied for diagnostic and vaccine development for a range of human and animal diseases.