Developing and expanding the bacterial glycotoolbox for animal pathogens

Lead Research Organisation: London School of Hygiene & Tropical Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Millions of animals die each year because of bacterial diseases. Despite this, and the fact that bacterial infection causes significant losses to the farming industry, the technologies designed to protect humans against bacterial infections have often not been available to combat animal disease. The biggest reason for this is cost. This proposal aims to harness genes from a bacterium that causes significant sickness and mortality in pigs. In preliminary experiments we discovered two genes from a pig pathogen called Actinobacillus pleuropneumoniae and found that they were responsible for making a sugar structure (polysaccharide) on the surface of this bacterium. We have been able to transfer these genes into a safe laboratory strain of Escherichia coli and to make a first generation of vaccine. This vaccine is based on the linkage of a protein to this sugar component. Crucially, the genes coding for this polysaccharide are present in all A. pleuropneumoniae strains. We believe that this may be a chink in the armour of this pig pathogen and would like to generate several vaccine formulations to test this theory. The method used to make this type of vaccine, called a glycoconjugate, is much cheaper than traditional techniques, which have prevented their widespread use in animals.
Our interests will also go beyond using the genes to make a vaccine against A. pleuropneumoniae. We are also going to explore if the protein responsible for making this vaccine has potential to be used as a tool to unlock current technological limitations. And make possible the assembly of novel vaccine formulations.

Technical Summary

For the past decade we have been developing an in vivo glycosylation method in Escherichia coli termed protein glycan coupling technology that expedites the production of pure inexpensive glycoconjugate vaccines. The method relies on the cloning of a polysaccharide coding region into a safe laboratory strain, followed by the introduction of an acceptor protein. The protein and glycan are coupled by the oligosaccharyltransferase enzyme PglB. The glycoconjugate can then be purified directly from the cultured E. coli in a single step procedure. However, PglB has limitations.

PGCT has now been clearly established for the production of glycoconjugate vaccines against Shigella dysenteriae, Francisella tularensis, Staphylococcus aureus and pathogenic E. coli. However, its potential application for animal pathogens is unexplored. There are limitations to PGCT and these mean that it has yet to be developed for veterinary purposes. This proposal will exploit a newly discovered N-linked glycosylation system that the applicants have characterised from the pig pathogen Actinobacillus pleuropneumoniae that promises to overcome the current limitations of the PgB-based system currently used in PGCT. Firstly novel glycoconjugate vaccine formulations will be developed against A. pleuropneumoniae which have the potential to protect against all serotypes. In addition, multivalent vaccines will be assembled by transferring the genes used to make the A. pleuropneumoniae polysaccharide specific component into other pig pathogens, Haemophilus parasuis and Streptococcus suis.

Finally the biotechnological potential of NGT will be investigated, these experiments will ascertain if NGT is a suitable tool for glycoconjugate production and have the potential to further broaden the applicability of PGCT, hence generating a novel biotechnological tool that will facilitate a novel vaccine assembly mechanism.

Planned Impact

The studies described in this proposal aim to generate a novel glycoconjugate vaccine and vaccine formulations against A. pleuropneumoniae. Harnessing this knowledge and in combination with our understanding of transposon mutagenesis, will also enable the first generation of multivalent vaccines by creating H. parasuis and S. suis strains capable of making the A. pleuropneumoniae specific glucose polymer within their cytoplasmic compartments.

The potential impact of an A. pleuropneumoniae vaccine capable of protecting against all serotypes alone is considerable, but the possibility of enhancing this vaccine strategy by combining it with a live attenuated strain of H. parasuis and S. suis is appealing. Through our collaboration with Professor Markus Aebi's group at ETH University, Zurich, we have been able to test the glycosylation status of proteins expressed in preliminary experiments. We have also been contacted by Malcisbo, rated 14th out of the top 100 start up companies in Switzerland. We have held lengthy discussions regarding the potential of an NGT-based glycoconjugate, and are in the process of setting up an agreement to share knowledge and genetic tools.

The second aspect of this proposal, understanding the biotechnological capability of NGT to overcome the limitations of PglB based technologies, is of particular relevance to this funding call. Should the proof-of-principle study using the S. suis capsular polysaccharide locus be successful, it could lead to the utilisation of this novel enzyme in order to tackle vaccine generation against a wide variety of pathogenic organisms for both animals and humans. To use Streptococcus pneumoniae as an example, over 90 capsule types exist but only a handful are substrates for PglB whilst the majority have glucose as their starting sugar.

As the leading group worldwide in the use of Protein Glycan Coupling Technology (PGCT), we are in frequent contact with industrial partners. The Francisella tularensis glycoconjugate vaccine generated at the LSHTM was assembled using an acceptor protein from Glycovaxyn. A Swiss spin off founded from the discovery of PglB and now the lead company in this method of biological conjugation.

Glycovaxyn and other companies with particular interest in animal vaccinology such as Zoetis, Merck and Malcisbo should provide a way to take the vaccines generated from this research to commercially viable products.

The potential impact of the research will also be realised through dissemination of our studies by publication in international peer-reviewed journals (where the applicants have a strong record) and by poster and oral presentations at major national and international meetings. The applicants have a track record of communicating the results of their research to the public (e.g. school children) and the mass media (radio broadcasts, Sky, ITV and BBC broadcasts). We also will maintain our international links in PGCT development with the Aebi and Syzmanski groups with whom we have long standing collaborations.

Exploitation of our research will continue to be delivered though the LSHTM technology transfer office, with whom the principal applicants have strong links. Two patents based on our research and PGCT has been filed and preliminary discussions have been made regarding potential licensing agreements and these will continue as and when appropriate opportunities arise.

In summary, we have demonstrated our capabilities of developing and nurturing our research relating to PGCT. We have wide collaborations internationally in both basic research laboratories and companies ranging from fledgling SME such as Malcisbo and big pharmaceutical companies such as Pfizer.

Publications

10 25 50

 
Description The current impacts of the research are that several patents have been developed through the research funded on this grant. Novel genetic tools and testing has now begun to understand the true applicability to the central enzyme in this application called NGT. My interest in veterinary vaccinology has grown and the veterinary field is becoming more aware of the glycobiology related research that I do. I have also taken an interest in the potential biotechnological application of the latest glycoengineering discoveries and applied in 2017 for a BBSRC/RSE Fellowship. I was awarded this I year fellowship in September 2017 and it is giving me the time and the funds to travel globally and talk to industry about how to make my work as industrially relevant as possible as well as learn about the process of commercialising research. I have been fortunate enough to have been given time to spend with some of the largest pharmaceutical companies and have discussions around how diseases are selected as targets for vaccine development, what the unanswered questions are regarding particular diseases, and how the basic research that we do could be slightly improved in order to make the most impact.
Exploitation Route The genetic tools to allow new vaccine candidates to be generated have been successfully assembled. This material led to a meeting wtih a major pharmaceutical company who has shared confidential data and materials in order to enable preliminary testing of our vaccine candidates in vitro. Now that a follow on award has been granted, we will aim to move vaccine candidates forward with our industrial partner.
Sectors Agriculture

Food and Drink

Environment

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description The research and specifically the development of a candidate A. pleuropneumoniae vaccine, has contributed significantly to discussions with several veterinary vaccine companies. We have secured a letter of support from Boehringer Ingleheim (BI) in September 2017 to apply for further grant funding in order to collaborate with industry on the development of a vaccine against porcine pleuropneumoniae. In March 2018, I visited BI and presented our latest glycoengineering research to their team investigating porcine bacterial disease. We will be applying for a follow on grant in April 2018 to develop the vaccines further and test them in industrially relevant models. As a new update, the follow on grant was awarded and is due to begin on the 1st of April 2019.
First Year Of Impact 2018
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Developing the E. coli GlycoCell
Amount £374,467 (GBP)
Funding ID BB/R008124/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 04/2021
 
Description Development, production and testing of novel glycoconjugate pig vaccines
Amount £557,778 (GBP)
Funding ID BB/S004963/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 12/2021
 
Description RSE/BBSRC Enterprise Fellowship
Amount £39,324 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 09/2018
 
Description Responsive Mode Vet Vaccines
Amount £5,303,258 (GBP)
Funding ID BB/N001591/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 03/2021
 
Description The Bloomsbury SET
Amount £333,604 (GBP)
Funding ID CCF17-7779 
Organisation Royal Veterinary College (RVC) 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 03/2021
 
Title Apx toxin fragment glycosylation 
Description One of the outcomes of this project is the assembly of plasmids designed to combine the N linked glycosylation system from Actinobacillus pleuropneumoniae and Apx toxin fragments. The NGT/AGT glycosylation operon was moved onto the chromosome on a safe laboratory strain of E. coli, generating a stable integrant mutant capable of glycosylating any acceptor protein with the glucose epitope made by Actinobacillus pleuropneumoniae itself. The plasmids assembled during this award encode for fragments of all of the Apx toxins and enable the attachment of a glucose to generate a prototype glycoconjugate vaccine. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact We are currently in contact with a pharmaceutical company to arrange for testing of the prototype vaccine that we have generated. However, details of the company and the testing method proposed are currently confidential. 
 
Title NGT/AGTminiTn5 
Description A miniTn5Zeo transposon was modified to carry the genes coding for the N linked glycosylation system from Actinobacillus pleuropneumoniae. This genetic tool has been used to successfully integrate the agt/ngt operon into the chromosome of laboratory E. coli strains. These strains are currently being used to generate prototype glycosylated Apx toxins but could be extended to allow cytoplasmic glycosylation of any protein. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact The genetic tool developed is allowing for the assembly of novel vaccine combinations in order to attempt to protect pigs from Actinobacillus pleuropneumoniae. 
 
Title Additional file 1: of Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines 
Description Table S1. Full LC-MS/MS output. (XLSX 15 kb) 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_1_of_Cytoplasmic_glycoengineering_of_Ap...
 
Title Additional file 1: of Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines 
Description Table S1. Full LC-MS/MS output. (XLSX 15 kb) 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_1_of_Cytoplasmic_glycoengineering_of_Ap...
 
Description AW 
Organisation Animal Health Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are in the process of genetically modifying a strain generated by our collaborator in order to produce what we hope will be a multi valent vaccine to protect horses against strangles and pneumonia caused by S. pneumoniae. We are employing the knowledge that my research team has accumulated in glycobiology to add value to an existing patented vaccine generated by our collaborator at the Animal Health Trust.
Collaborator Contribution The collaborator has provided the base strains under an MTA for us to work on the modification to generate a multivalent vaccine. The collaborator is a know expert in the field of genetic modification of S. equi and has also provided invaluable knowledge on this organism.
Impact No outputs yet but we hope to generate preliminary data to apply for a joint grant proposal in the near future.
Start Year 2016
 
Description BI 
Organisation Boehringer Ingelheim
Country Germany 
Sector Private 
PI Contribution Industry collaboration in order to identify if our vaccine candidate prototypes could be useful. A letter of support was issued in Sept 2017 for a follow on grant application with a deadline of April 2018.
Collaborator Contribution BI have provided a letter of support and if the follow on grant application is successful, they have committed to providing industrially relevant testing of vaccine candidates.
Impact N/A as currently this is a support letter for a future grant proposal.
Start Year 2017
 
Description Royal Veterinary College Bloomsbury SET 
Organisation Royal Veterinary College (RVC)
Country United Kingdom 
Sector Academic/University 
PI Contribution We utilised our knowledge of biological glycoengineering to formulate a workplan in the generation of a whole cell and glycoconjugate vaccine prototype.
Collaborator Contribution We have co-applied for a Bloomsbury SET grant . Awarded by Research England. In order to develop and immunologically appraise a vaccine against the respiratory pathogen Streptococcus suis. Our co-applicant, Professor Dirk Werling will develop assays in order to measure and observe the immunological response from pig lung tissue to exposure to the capsular polysaccharide of S. suis. In vitro organ culture models will be set up at the RVC and all immunological characterisation of candidate vaccines produced at the LSHTM will be tested at the RVC.
Impact This partnership is due to officially commence on the 1st of April 2019. It is a multidisciplinary approach in that it will utilise expertise in the following fields: Molecular Microbiology Glycoengineering/Glycobiology Immunology
Start Year 2018
 
Title Oligosaccharyltransferase Polypeptide 
Description The disclosure relates to an oligosaccharyltransferase polypeptide and its use in the synthesis of glycoconjugates in bacterial cells; vaccines and immunogenic compositions comprising said glycoconjugates and their use 5 in the prevention and/or treatment of bacterial infection. 
IP Reference GB1603948.9 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This patent describes the discovery and the demonstration of activity of a new PglB from the bacterium Campylobacter sputorum. This PglB is likely to have commercial implications as it bares very little similarity to the already licensed C. jejuni PglB that all other glycoengineering work is based on.
 
Title Whole Cell Vaccines 
Description The disclosure relates to attenuated bacterial cells expressing glycan and glycoconjugate antigens and their use in the manufacture of whole cell vaccines or immunogenic compositions effective at eliciting an immune response in non-human animal species and preventing or treating bacterial infections. 
IP Reference GB1603958.8 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This is a patent demonstrating how licensed live attenuated vaccines can be modified to carry either glycoengineering tools to make and deliver subunit vaccines or for surface expression of foreign antigens. This application should have significant implications in the development of novel vaccine combinations for both veterinary and human vaccinology.
 
Title Whole Cell vaccine 
Description The disclosure relates to attenuated bacterial species having deletions and/or mutations in genes that render the bacterial species viable but unable to invoke a pathogenic or deleterious effect on an infected subject, for example a human or a non-human animal subject; and including vaccine or immunogenic compositions comprising the attenuated bacterial species with methods of vaccination and immunisation. 
IP Reference GB1603963.8 
Protection Patent / Patent application
Year Protection Granted 2016
Licensed Yes
Impact This patent refers to the generation of live attenuated Streptococcus suis strains. These may be modifed for heterologous surface expression and could lead to novel vaccines.
 
Company Name ArkVax 
Description ArkVax develops vaccines for animals through glycoengineering. 
Year Established 2020 
Impact N/A as the company is currently in the process of securing contracts and funding
Website https://www.arkvax.com/
 
Description Attendance at the Royal Veterinary College Research club and presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at the RVC veterinary research club where I presented on the glycoengineering work that I am involved with. Specifically for the generation of animal vaccines. This led to much debate and I was approached by several research groups to be invited to talk at their institues as well as set up future potential collaborations.
Year(s) Of Engagement Activity 2015
 
Description LSHTM-BRI Institut Pasteur workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A meeting was held between the Institut Pasteur and the LSHTM-BRI with the aim of demonstrating the work the two insitutes are doing in relation to anti microbial resistance and vaccine development
Year(s) Of Engagement Activity 2015
 
Description Presentation of technology to Riga Institute for Medical Research Leuven 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Presented at a meeting between LSHTM and the RIGA institute Leuven. The idea of this meeting was to see if we could find areas of mutual interest between the 2 institutes. I discussed the human and veterinary research that we are undertaking at the LSHTM.
Year(s) Of Engagement Activity 2019
 
Description Seminar GSK Siena November 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presented our glycoengineering capabilities to Rino Rappuoli's team at GSK Siena in November 2017. This led to interesting discussions around the subject of vaccine design and gave us an opportunity to begin to think about how we could modify our research projects to work alongside the latest findings from GSK.
Year(s) Of Engagement Activity 2017
 
Description Talk at Vaccine Centre 2019 retreat 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I presented at the LSHTM Vaccine Centre bi-annual meeting. The title of the talk was 'A new approach to vaccine development through an industry-academia alliance'. The objective of the talk was to open a discussion as to how important it is for vaccine researchers who are developing new technologies to interact with industry during the development phase of their work, in order to avoid mistakes, such as incorrect target design or developing unscalable systems. I highlighted the benefits and the potential downside of such collaborations based on personal experience.
The audience consisted of LSHTM vaccine centre members from Gambia and London as well as Oxford University and Imperial University. The audience was a mixture of principal investigators, post docs and PhD students.
Year(s) Of Engagement Activity 2019
URL https://vaccines.lshtm.ac.uk/events/past-events/retreats/
 
Description Uk Vaccine Network working group 2 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact I was involved in a working group of 10 vaccinology experts that met to discuss the UK preparedness to deal with for a potential new pandemic caused by an unknown organism. My role was to represent bacteriology and vaccine development for bacterial pathogens.
Year(s) Of Engagement Activity 2015
 
Description Vaccine Centre Retreat 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attended and presented at the first LSHTM Vaccine centre retreat. A group was assembled consisting of lab scientists all the way through to epidemiologists and invited guests who were policy makers at the level of GAVI and WHO. This meeting led to discussions regarding all aspects of vaccination, from funding to the potential future of vaccine research. This has led to requests for me to become part of the LSHTM vaccine centre working group and getting involved in arranging further meetings as well as topics of discussion.
Year(s) Of Engagement Activity 2016