Bacterial oligosaccharyltransferase for glycoengineering and vaccine development

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Vaccination has been incredibly successful in reducing the burden of infectious diseases. Examples of successful vaccines include those against the deadly bacteria Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. The basis for these vaccines is a complex sugar structure, known as the capsule, which covers bacterial cells protecting them from immune attack. In order to evoke an appropriate immune response, vaccines against these bacteria consist of capsule linked to a protein carrier forming a glycoprotein or glycoconjugate. However, despite the success of these glycoconjugate vaccines they have major drawbacks in terms of the technical difficulties in purifying the capsule material from bacterial cells and then conjugating the capsule to carrier proteins. Additionally, capsules are often highly variable, and the specific immunity elicited by immunisation with one type of capsule will not protect against bacteria with different capsule structures. Thus as new disease strains emerge (e.g. from selective pressure by large scale vaccination regimes) the existing vaccines become ineffective. An inexpensive, rapid and flexible method for glycoconjugate vaccine production would enable a more effective response to the emergence of new pathogenic bacterial strains with different capsule structures. One such approach is to produce glycoconjugate vaccines in the genetically tractable bacterium Escherichia coli. E. coli is already used as a 'cellular factory' to produce large amounts of proteins; however, until recently it has not been possible to generate glycoproteins in this bacterium. That could now change. We have recently identified and characterised a gene cluster (pgl) which is responsible for the synthesis of glycoproteins in the bacterial food-borne pathogen, Campylobacter jejuni. We have been able to transfer the segment of DNA containing the pgl genes into E. coli to produce recombinant glycoproteins, thus opening up the field of glycoengineering. The key enzyme in the C. jejuni pathway that couples proteins to sugars is the oligosaccharyltransferase protein termed CjPglB. Although CjPglB can transfer many sugar structures unfortunately there are many structures from various pathogens that it cannot. Essentially, the end of the glycostructure that is attached to the protein by CjPglB, must have a sugar unit with a specific configuration - an acetamido group at the C-2 position of the sugar at the reducing end of the glycan. This severely limits the potential applications of this technology. Indeed many capsules of pathogenic bacteria do not have this configuration and therefore CjPglB could not be used to produce glycoconjugate vaccines for protection against these bacteria. In this study we propose a number of strategies to overcome this problem. We will seek to identify or engineer alternative PglB proteins that will have a modified specificity for different glycostructures. We will use a dual approach of seeking alternative PglBs from other bacteria that may naturally have a different specificity to the original CjPglB, and also a mutagenesis approach to alter the enzymatic specificity of CjPglB. To ascertain if the specificity of the natural and mutated PglBs have been altered we will test separate capsular polysaccharides from the important pathogens Streptococcus pneumoniae and Burkholderia pseudomallei to determine if the respective capsules can now be coupled/conjugated to an appropriate carrier protein. The new recombinant glycoconjugates in E. coli will be ideal vaccine candidates that can be readily purified and tested. The glycoengineering principles to be pioneered in this study could be applied generically to the design of other glycoconjugate and combination vaccines. Irrespective of vaccine development, this new and emerging technology will be of direct importance to scientists interested in basic research and in applied research in glyco-biotechnology.

Technical Summary

Glycan-containing biomolecules are ubiquitous and although involved in diverse processes ranging from immune recognition to cancer development their significance remains underestimated. Furthermore, and in contrast to polypeptides and nucleic acids, glycoproteins have escaped biotechnological applications. However our demonstration of glycoprotein biosynthesis in the model bacterium Escherichia coli, through the activity of a novel C. jejuni oligosaccharyltransferase (CjPglB), is a significant advance (Wacker et al, Science 2002). Recently, along with our collaborators we have demonstrated that CjPglB has a relaxed specificity enabling transfer of diverse polysaccharides, including lipopolysaccharide and capsules, to proteins with appropriate D/E-X-N-Z-S/T consensus sequons. However, CjPglB is unable to transfer all glycans; one requirement is for an acetamido group at the C-2 position of the reducing end sugar of the glycan. Thus before the production of diverse protein-polysaccharide conjugates in E. coli can be fully realised, a wider specificity of the PglB enzyme is required. To this end, we propose a dual strategy of mining further bacterial pglB orthologues combined with directed evolution approaches to explore the specificity range of characterised oligosaccharyl transferases. Additionally, we will attempt structural characterisation of CjPglB to provide mechanistic information on oligosaccharide transferase activity. As test cases we will determine if capsules from bacterial pathogens Streptococcus pneumoniae and Burkholderia pseudomallei, that lack an acetamido group at the C-2 position, can be transferred to an appropriate acceptor protein by the novel or modified PglBs. If successful these studies could radically improve the production and range of novel protein glycoconjugates for vaccines. Irrespective of vaccine development, we are convinced that the studies will be important for the development of glycoengineering in both basic and applied research.

Publications

10 25 50
 
Description We have developed Protein Glycan Coupling Technology (PGCT) as a method to produce recombinant glycoconjugate vaccines in E. coli. In this study, we identified new enzyme which make PGCT more efficient. The main application of PGCT is in the production of low cost recombinant glycoconjugate vaccines.
Exploitation Route Protein Glycan Coupling Technology (PGCT) as a method to produce recombinant glycoconjugates molecules in E. coli is now regularly used in academia and industry for multiple glycoengineering purposes. For example, in the vaccine industry used by GlycoVaxyn (recently purchased by GSK) and VaxAlta. The principles of PGCT have been used as a platform technology to initiate a Spin Out company ArcVax at the LSHTM.
Sectors Manufacturing, including Industrial Biotechology

 
Description In this research program we were able to 1) efficiently express bacterial glycans such as O-antigens in E. coli and 2) transfer the O-antigen to a carrier protein in E. coli using novel oligosaccharyltransferase. This has helped to establish Protein Glycan Coupling Technology (PGCT) as a method to produce recombinant glycoconjugate vaccines in E. coli. This has potential in the vaccine industry used by GlycoVaxyn (recently purchased by GSK) and VaxAlta. The principles of PGCT have been used as a platform technology to initiate a Spin Out company ArcVax at the LSHTM.
First Year Of Impact 2010
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
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 04/2020 
End 04/2022
 
Description Developing and expanding the bacterial glycotoolbox for animal pathogens
Amount £200,000 (GBP)
Funding ID BB/M01925X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 09/2017
 
Description Developing the E. coli GlycoCell
Amount £455,000 (GBP)
Funding ID BB/R008124/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 06/2021
 
Description Gates Grand Challenge
Amount $1,000,000 (USD)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 03/2011 
End 03/2014
 
Description Glycoengineering of Veterinary Vaccines
Amount £5,300,000 (GBP)
Funding ID BB/N001591/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 04/2021
 
Description Institutional Translation Partnership Awards 'London School of Hygiene & Tropical Medicine'
Amount £800,000 (GBP)
Funding ID 214227/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2019 
End 03/2021
 
Description Universal protection against Streptococcus pnuemoniae
Amount £989,000 (GBP)
Funding ID MR/R001871/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 04/2021
 
Description Wellcome Trust Senior Investigator award
Amount £2,100,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2014 
End 04/2019
 
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 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 Vaccine development for defence purposes 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution New vaccine technology platform for making glycoconjugate vaccines against Burkholderia pseudomallei and Francisella tulerensis
Collaborator Contribution Technology know how, specific animal testing facilities
Impact Several vaccine candidates tested and some may go forward to vaccine trials
 
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 Bacterial Glycotoolbox 
Description Fine tuning of the E. coli cell to express and produce bacterial glycans 
IP Reference GB1603963.8 
Protection Patent granted
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact The establishment of vaccine technology platform to engineer low cost recombinant glycoconjugate vaccines
 
Title Glycoconjugate Vaccines 
Description Novel method to construct combinations of glycoconjugate vaccines 
IP Reference WO4307P 
Protection Patent granted
Year Protection Granted
Licensed No
Impact Can cheaply produce novel glycoconjugate vaccines for humans and animals
 
Title Glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen 
Description New method for constructing glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen 
IP Reference WO2018046955 
Protection Patent granted
Year Protection Granted 2015
Licensed Commercial In Confidence
Impact Method to make vaccines against Francisella tularensis for which there is no current human vaccine
 
Title MAGIC Mobile-element Assisted Glycoconjugate Insertion on Chromosome 
Description A method to improve glycosylation of proteins 
IP Reference US20150344928 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact A method that could produce inexpensive glycoproteins
 
Title Recombinant Protein Production 
Description The use of Protein Glycan Coupling Technology to produce low cost recombinant vaccines. In this example against Francisella tulerensis 
IP Reference GB1606036.0 
Protection Patent granted
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact The establishment of vaccine technology platform to engineer low cost recombinant glycoconjugate vaccines
 
Description Broadcast interviews (Newsnight, Panorama, One Show) 
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 Public/other audiences
Results and Impact Interest in research at LSHTM

Better understanding of science
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014
 
Description TV Broadcasts 
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 BBC Breakfast TV interview on vaccine design and antimicrobial resistance
BBC one interview on Campylobacter in Food chain
One Show BBC on antimicrobial resistance
Year(s) Of Engagement Activity 2015