Developing the E. coli GlycoCell

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Department of Pathogen Molecular Biology

Abstract

Vaccines are a critical component of defence against infectious disease in both humans and animals. Large scale vaccination has eliminated some of the most dangerous diseases that have faced humanity.
Polysaccharides or glycans are complex sugar based structures that are central to everyday life and the biotechnology industry. In contrast to the cloning revolution for DNA and protein molecules, the cloning, expression and characterisation of glycan-based molecules is in its infancy. This is due to the complexity of the structures and difficulties in their purification and production in a simple system that faithfully reproduces the molecules in sufficient yield.

Polysaccharides are large chains made up of sugars that are often unique to each species of bacterium. They can be found in an almost infinite variety of structures, most of which remain to be characterised. In addition, the sugar chains often coat the outside of the bacterial cell, and are readily detected by the human immune system. These sugar coats therefore make excellent vaccines: they will activate the immune system, which will then detect and respond to an infection by the relevant bacteria much more effectively. The sugar coats make even more effective vaccines if they can be attached to other components of the bacteria such as proteins. This provides multiple triggers for the immune system, and increases the lifetime of the body's immune response to the sugar coat.

This project will develop a system to efficiently produce bacterial polysaccharides and polysaccharide-protein combinations that make effective vaccines. A major reason why these sugar coats are not used for vaccines against a wider range of bacteria is that they are often difficult to prepare and to attach to other cellular components, rendering the manufacturing process expensive. Our system will overcome these problems by engineering a safe laboratory bacterium (E. coli) to act as a mini-cell factory and efficiently make the sugar coat. We will use a recently discovered enzyme that will physically link the sugar coat directly to another bacterial component (protein): this reduces the complexity of preparing the vaccine considerably, thereby lowering manufacturing costs.

To achieve these goals, we will firstly take a common E. coli bacterium, and remove its own sugar coat components using genetics. This will ensure that the entire product from the system is the desired vaccine. We will then add the components required to make the desired sugar coat: these will consist of genes needed to make individual sugar units, and genes that link these individual units together to make long chains of sugar. We will then engineer into the bacterial cell the ability to attach the sugar coat to other bacterial components (e.g. proteins). As a testing ground, to develop our platform technologies, we have chosen the cloning and expression of several Streptococcus pneumoniae variant capsular polysaccharides. S. pneumoniae is a major pathogen responsible for 14.5 million annual infections worldwide and >800,000 deaths in children under 5 years of age. S. pneumoniae is not just an important global pathogen, it is an ideal model to study for our tailored engineering approach due to the variation in glycostructures present with over 90 different capsular polysaccharides. We will compare the effectiveness of our approach at each stage with our existing technology to efficiently make recombinant S. pneumoniae glycoconjugate vaccines.
The efficient cloning and production of polysaccharides in these newly generated E. coli strains promises to break new ground in biotechnological applications requiring the efficient production of polysaccharides or polysaccharide complexes, including making glycoconjugate vaccines. Finally, the knowledge obtained during the project will be invaluable to help educate the scientific community on how to repurpose an E. coli cell for optimal sugar assembly and production.

Technical Summary

Bacteria express the most diverse array of glycostructures among living organisms which includes capsules, LPS, O- and N-linked glycans, most of which remain to be characterised. In bacteria, these structures are frequently encoded in operons that can be readily cloned into E. coli cells. We have developed Glycan Expression Technology (GET), where such gene cassettes are expressed in an E. coli host for the efficient production of glycostructures. In addition, we have pioneered Protein Glycan Coupling Technology (PGCT) where an expressed glycan can be coupled to a given protein, using bacterial oligosaccharyltransferases, in an E. coli host cell. We will produce glyco-tailored E. coli strains for the efficient expression of glycans and glycan/protein combinations. We will: (i) "tailor" the E. coli cell to produce only defined nucleotide activated sugar precursors to reduce metabolic load; (ii) add glycobiosynthetic capabilities that are lacking and integrate these genes stably on the E. coli chromosome; (iii) remove factors from E. coli which interfere with correct glycan assembly, efficient polymerization and purification, including reducing endotoxicity. We will develop a systematic approach to monitoring glycan expression by using iTRAQ to measure proteome changes and flux balance analysis to identify potential bottlenecks in metabolism that could be removed or alleviated to produce needed precursors more effectively. Glyco-tailored strains will be assessed for improved glycoconjugate vaccine production using several clinically relevant Streptococcus pneumoniae serotypes. Improvement of GET and PGCT would have extensive benefits for applications in glycan production, synthetic biology and glycoengineering, including the production of glycoconjugate vaccines and humanised glycoproteins. However, to achieve this a fundamental understanding of the expression of foreign glycostructures in E. coli, together with the development of a new bank of strains is required.

Planned Impact

In this programme of research the applicants will exploit a number of recent discoveries and technical innovations, many of which have been developed in the applicants' laboratory through BBSRC support. These include the characterisation of several polysaccharide coding regions and of novel glycosylation systems, the production of recombinant glyco-modified proteins, glycoengineering for the production of vaccines, development of the glyco "tool box" and "synthetic glycobiology" (using gene cassettes for known glycostructures and the production of a new set of characterised E. coli strains). Further validation and development of this methodology will benefit the wider scientific research community as a technology platform for numerous glycoengineering applications in the laboratory sciences, healthcare services and biotechnology-based industry. For example, the newly engineered E. coli strains could be used as safe sources of Streptococcus pneumoniae capsular polysaccharide, not just for protein glycan coupling technology, but also for whole cell studies or outer membrane vesicles, or for standard chemical coupling based technologies.

The platform that we will generate will allow a much wider range of polysaccharides to be manufactured, and an important aim of our work will be to demonstrate that this can be achieved in quantity. Our program will firstly offer new polysaccharides and vaccine candidates for public health pathogens such as Streptococcus pneumoniae. This will assist in the development and manufacture of new vaccines, and this will provide significant benefits to the UK economy. Vaccines, in particular, are proven for the control of infectious diseases in both humans and in animals, and suitably designed vaccines will reduce our reliance on antibiotics. In the long term, the program of work will benefit the health and wealth of the nation, and the delivery of low cost glycoengineered vaccines will have benefits worldwide.
 
Description Improved E. coli strains to increase the expression and production of glycoconjugate vaccines.
Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse.
In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences.
In conclusion, amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4x and 14x more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community.

This bank of strains has proven useful in the development of pneumococcal glycoconjugate vaccine, poultry vaccines and several other recombinant glycoconjugate vaccines.
Exploitation Route Strains would be useful for both academia and industry for expressing recombinant glycans
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description A bank and family tree of over 15 strains have been produced that have improved the expression of glycan in E. coli and for the Protein Glycan Coupling Technology for the production of recombinant glycoconjugate vaccines including pneumococcal and poultry vaccine candidates
First Year Of Impact 2020
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Bloomsbury SET Research England Development of Streptococcus suis vaccines
Amount £310,000 (GBP)
Organisation Royal Veterinary College (RVC) 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 04/2021
 
Description Developing a multivalent Streptococcus pneumoniae recombinant glycoconjugate vaccine for preventing meningitis'
Amount £220,000 (GBP)
Organisation Meningitis Now 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 04/2024
 
Description Development and application of an Advanced Glycan Production Platform
Amount £447,107 (GBP)
Funding ID BB/W006146/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2022 
End 01/2025
 
Description Development and testing of a Streptococcus suis glycoconjugate vaccine
Amount £550,000 (GBP)
Organisation Research Councils UK (RCUK) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2021
 
Description Development of a Universal Group A Streptococcus Glycoconjugate Vaccine
Amount £403,000 (GBP)
Funding ID 221589/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2021 
End 01/2023
 
Description Development, production and testing of novel glycoconjugate pig vaccines
Amount £600,000 (GBP)
Funding ID BB/S004963/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 02/2021
 
Description Reducing the health & economic burden of Campylobacter using a live vaccine
Amount $600,000 (AUD)
Funding ID LP190100114 
Organisation Australian Research Council 
Sector Public
Country Australia
Start 09/2021 
End 10/2024
 
Description The Future Vaccine Manufacturing Research Hub (Vax-Hub)
Amount £7,000,000 (GBP)
Funding ID EP/R013756/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 10/2022
 
Description The GCRF One Health Poultry HUB
Amount £20,000,000 (GBP)
Organisation Research Councils UK (RCUK) 
Sector Public
Country United Kingdom
Start 02/2019 
End 02/2024
 
Description The production of "Shigella plus" low-cost recombinant Shigella glycoconjugate vaccines'
Amount £470,213 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2022 
End 02/2024
 
Description Unravelling the molecular mechanisms of invasive pneumococcal disease
Amount £1,900,000 (GBP)
Funding ID 221803/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2021 
End 01/2026
 
Description Glycoengineering pathways in E. coli 
Organisation University of Arkansas Medical Center
Country United States 
Sector Hospitals 
PI Contribution We have supplied 11 E. coli strains derived from our E. coli GlycoCell project to evaluate for efficiency fro glycosylation and to improve glycol yields based on reshunting UndeCPP pathways.
Collaborator Contribution In collaboration with Professor Matthew Jorgenson, UAMS we are investigating the role of recycling UndeCPP in the efficiency of glycoengineering in E. coli cells
Impact Improved E. coli strains for glycoengineering
Start Year 2020
 
Description Hilleman Labs 
Organisation MSD Wellcome Trust Hilleman Laboratories
Country India 
Sector Charity/Non Profit 
PI Contribution Intellectual contribution and vaccine development and production.
Collaborator Contribution Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Description Merck MSD 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution Collaboration on vaccine development
Collaborator Contribution Intellectual contribution and vaccine development and production
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Description Testing of Brucella vaccine candidates 
Organisation Rajamangala University of Technology Srivijaya
Country Thailand 
Sector Academic/University 
PI Contribution Provision of Brucella vaccine candidates
Collaborator Contribution Testing of Brucella vaccine candidates
Impact Still early stages
Start Year 2021
 
Description Vabiotech vaccine production 
Organisation VabioTech
Country Viet Nam 
Sector Public 
PI Contribution Intellectual contribution and vaccine development and production.
Collaborator Contribution Intellectual contribution and vaccine development and production.
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Description huvepharma 
Organisation Huvepharma
Country Bulgaria 
Sector Private 
PI Contribution Intellectual contribution, vaccine development and production.
Collaborator Contribution Intellectual contribution, vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Title Oligosaccharyltransferase Polypeptide 
Description New enzyme to improve glycoengineering in E. coli 
IP Reference GB1704103.9 
Protection Patent application published
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact Improve glycoengineering in the E. coli cell
 
Title THERAPEUTIC FOR TREATING CLOSTRIDIUM DIFFICILE INFECTION 
Description The invention relates to deoxyribonuclease for use in the treatment of a suspected or existing C. difficile infection; a pharmaceutical or veterinary composition or formulation comprising at least deoxyribonucleasefor use in the treatment of a suspected or existing C. difficile infection; a combination therapeutic comprising at least deoxyribonucleasefor use in the treatment of a suspected or existing C. difficile infection; a method of treating a mammal suspected of being infected with, or infected with, C. difficile comprising the use of at least deoxyribonuclease; a method of cleaning or sterilising a material or product comprising the use of at least deoxyribonuclease; and a cleaning or sterilising product impregnated with or containing at least deoxyribonuclease. 
IP Reference WO2013175172 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact Simple treatment to reduce C. difficile disease
 
Company Name ARKVAX LIMITED 
Description A new spin out company that derived from ArcVax was formed on 20/10/2020. The company won a competitive accelerator award and currently has labs at Babraham Research Park, Cambs and focuses on using novel glycoengineering technology for the development of glycoconjugate vaccines with a particular emphasis on animal vaccines 
Year Established 2020 
Impact The focus of the company is the development of multicomponent poultry, pig and ruminant vaccines. The business is supported contract research for vaccine candidate from established vet vaccine companies and further funding is currently being sought from a range of investors.
 
Company Name ArcVax 
Description A spin out company from LSHTM was set up in Dec 2016 to using Protein Glycan Coupling Technology to produce low cost glycoconjugate vaccines. Currently investment is being sought. 
Year Established 2016 
Impact To rapidly develop low cost candidate glycoconjugate vaccines, eg multiprotective vaccines for poultry.
 
Description 30+ TV interviews (BBC, SKY, Channel 4) on Covid vaccine delivery, production and use 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact 30+ TV interviews (BBC, SKY, Channel 4) on Covid vaccine delivery, production and use.
Received 100s of questions from general public many relating to vaccine confidence
Year(s) Of Engagement Activity 2020
 
Description 5 international TV interviews (eg BBC News, Swiss and Russian TV) on Covid vaccine delivery, production and use as well as SARS2-CoV epidemiology 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact 5 international TV interviews (eg BBC News, Swiss and Russian TV) on Covid vaccine delivery, production and use.
Received dozens of questions from general public many relating to vaccine confidence
Year(s) Of Engagement Activity 2020
 
Description 5 invited newspaper articles in Daily Mail TV interviews on Covid vaccine delivery, production and use. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 5 invited newspaper articles in Daily Mail TV interviews on Covid vaccine delivery, production and use.
Numerous response from general public who have felt reassured about taking Covid vaccine.
Year(s) Of Engagement Activity 2020