Glycoengineering of Veterinary Vaccines

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

Abstract

A healthily maintained livestock is essential for the economy and prosperity of the UK. Additionally some infected livestock are the source of human diseases, particularly through foodborne infections. Historically, vaccines have been the most successful and effective intervention to reduce the burden of infectious diseases in humans. By contrast, the application of vaccines in veterinary medicine is rudimentary, mainly due to the economic necessity for reduced costs to vaccinate animals and because our knowledge of the pathogens that cause animal diseases lags behind that of human counterparts.

A defining characteristic of a successful vaccine is the ability to evoke long-lasting protective immunity with minimal side effects. Many of the most successful human vaccines are glycoconjugates, a combination of a protein coupled to a glycan, which induces both a T-cell dependent and independent immune response generating a protective and lasting immunity. Examples of currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae, in which glycans (lipopolysaccharides or capsular polysaccharides) are chemically coupled to immunogenic carrier proteins. However, the production of these vaccines requires multistep procedures that are often complex and expensive, and can exhibit batch-to-batch variation.

We recently developed Protein Glycan Coupling Technology (PGCT) that can overcome the complex procedures required for chemically synthesising glycoconjugate vaccines by expressing the vaccine in an Escherichia coli cell in a single-step procedure. The advantages of applying PGCT to veterinary vaccines are (i) glycoconjugate vaccines can be produced at low cost, (ii) the flexibility of coupling "any glycan" with "any protein" facilitates the production of vaccine combinations providing the opportunity to evaluate a greater variety of vaccine candidates, and (iii) combination vaccines against more than one disease can be produced, further reducing cost and obviating the need to administer multiple vaccines (or antibiotics).

In this study we will use PGCT to produce inexpensive triple combination poultry vaccines to reduce infection from E. coli, Salmonella, Campylobacter jejuni/coli and C. perfringens. This will not only protect poultry flocks from severe disease but would also protect the human population from the most common foodborne infections including those caused by Salmonella and Campylobacter. In addition we will construct and evaluate a dual Coxiella/C. perfringens vaccine to protect cattle, sheep and goats against severe disease. This vaccine would also prevent the spread of Q-fever to humans, which is caused by the highly infectious Coxiella burnetii pathogen. The principles developed in this proposal could subsequently be widely applied to produce inexpensive efficacious vaccines against most animal species and promise to break new ground in veterinary vaccine production.

Technical Summary

An unmet need in veterinary vaccinology is the production of low cost effective vaccines that can protect against multiple infectious agents. We will aim to capitalise on our recent characterisation of a novel N-linked general glycosylation system in Campylobacter jejuni that can be used to engineer multiple combinations of glyco-modified proteins in different bacteria including E. coli and Salmonella species. This protein glycan coupling technology (PGCT) is proven in the production of human vaccines, but has yet to be applied to veterinary vaccines. We propose to use PGCT to construct dual and triple combination vaccines to reduce the carriage of Salmonella, Campylobacter, E. coli and Clostridium perfringens in poultry. The engineered constructs will also be used to investigate basic immunological responses in chickens to these pathogens, and will be tested for protection in chickens against infection.

To expedite the application of PGCT we will develop a more detailed understanding of glycobiosynthetic pathways in pathogenic bacteria including Coxiella burnetii. The novel LPS biosynthetic pathway will be thoroughly characterised by genetic, chemical and structural analyses. We will clone and express the LPS from C. burnetii in E. coli and couple this to genetic toxoids from C. perfringens including deactivated NetB to produce a dual vaccine. We will assess vaccine candidates produced by examining markers for humoral and cellular immunity, and the ability to induce protective immunity against C. perfringens toxins and C. burnetii in mice. In parallel with the development of the stated veterinary vaccines, we will use the opportunity to further innovate PGCT. We aim to improve the utility, efficacy and general applicability of PGCT for glycoconjugate vaccinology and for further glycobiotechnological applications. Additionally, we will foster our industrial collaborations to fully exploit the vaccines and innovations derived from this research.

Planned Impact

The economy
The knowledge generated in the program and application of the research would clearly benefit the poultry and livestock industry as well as farming communities. Ultimately, through reduced occurrence of food poisoning, the knowledge gained in this study will improve the health and wealth of the nation. The reduction of serious infections in livestock coupled with the development and manufacture of novel vaccines will provide significant benefits to the UK economy. The impacts of the research program are potentially enormous and manifold. Vaccines are proven for the control of infectious diseases in both humans and in animals, and suitably designed vaccines will reduce our reliance on antibiotics. With the UK livestock industry (including cows, pigs, sheep, poultry and fish) estimated to have an annual value of over £14bn in 2013, smart design vaccines will have direct benefits for the UK economy. Chickens alone are the world's most popular food animal with global poultry production tripling in the past 20 years and will continue to increase. Therefore, farmers and the agricultural industry will significantly benefit from cheaper more effective vaccines that target livestock.

The general public
The general public will benefit from less food poisoning in the reduction of C. jejuni and C. coli in the food chain, with the resultant economic benefit to the UK economy in terms of improved productivity. C. perfringens is major cause of disease in domesticated livestock ranging from enterotoxaemia in sheep, goals and calves to necrotic enteritis in poultry, a disease which is emerging following the EU ban on the use of antibiotics to promote growth. An effective poultry vaccine would also discourage the indiscriminate use of antimicrobials in livestock and contribute to reducing antimicrobial resistance. Thus a significant impact will be the reduction in antibiotic use, a key government policy and priority https://www.gov.uk/government/publications/uk-5-yearantimicrobial-resistance. Coxiella is a zoonotic agent therefore an effective animal vaccine would reduce transmission of Q fever to humans. Additionally, there would be a market for a human Coxiella vaccine to protect workers likely to come into contact with infected animals and where Q fever is endemic as well as for defense purposes. Therefore, the proposal will considerably enhance the quality of life and improve the economic competitiveness of the UK.

Academic and industrial organisations
The development of PGCT would enhance the commercial private sector for the production of vaccines and potentially for glycoengineering human therapeutics. We have close links with Zoetis, Merck, Glycovaxyn, VaxAlta and Malicisbo and will use licensing agreements through our respective technology transfer offices to ensure pipelines to vaccine production and exploitation are in place. Developing a basic understanding of the glycobiosynthetic pathways for the pathogens in this study will not only be important for understanding pathogenesis and vaccine production, but has other practical applications. The inhibition of bacterial glycosyltransferases is a useful target to disable the pathogenic bacteria providing a novel approach for antimicrobial development termed "glycobiotics". Additionally, bacterial glycans are often surface exposed and specific to individual species or virulent clones providing improved diagnostics benefitting human and veterinary health. The technology developed through may have enormous implications for policy makers to future disease outbreaks and impact on exports.

Training opportunities
The consortium will employ and train and develop a cohort of scientists with diverse experience with a "one health" mentality that can be applied in academia, the public sector and industry. The multidisciplinary team will add to the UK science base in an important and economically vital research area.

Publications

10 25 50
 
Description New live attenuated strains fro Salmonella and EPEC.

New Campylobacter and Clostridium perfringens poultry vaccines.

New improvements in bacterial glycoengineering (eg glycan expression, coupling and glycan seeking technology)
Exploitation Route Industrial collaborators such as Zoetis, Boehringer Ingelheim and Merck are interested in commercialising chicken and pig vaccines
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Objective (i) Construct, develop and test triple poultry vaccines against Escherichia coli, Campylobacter and Clostridium perfringens, and against Salmonella, Campylobacter and Clostridium perfringens. Toward subunit vaccines, we have cloned, expressed and validated the following proteins in E. coli harbouring the Campylobacter pgl locus, together with the cognate non-glycosylated control proteins; 1. Deactivated Pseudomonas exotoxin A dual glycan (to evaluate protection conferred by the C. jejuni heptasaccharide) 2. Deactivated Pseudomonas exotoxin A decaglycan 3. Deactivated Clostridium perfringens toxin NetB dual glycan (P4 in original proposal) 4. Campylobacter fibronectin-binding adhesin FlpA dual glycan 5. Campylobacter superoxide dismutase SodB triple glycan These are currently being tested in chickens In addition we have designed new live attenuated Salmonella ST4/74 and APEC chi7122 strains and engineered these to express the PGCT system. A major development has been our construction of the C. jejuni pgl locus using the Gibson Assembly process and the stable integration of this locus into the chromosomes of APEC chi7122 and ST4/74 by double recombination using a positive-selection suicide replicon. These stable pgl integrants are the ideal templates to introduce attenuating mutations. Attenuated strains will be further modified by expression of Campylobacter and/or Clostridium antigens. Sequencing and in vivo testing of the carriers is ongoing . With this innovation, alternative LAVs (including potentially the PoulVac strains) can be readily rendered amenable as multi-component glycoconjugate vaccines Objective (ii) Produce a dual Coxiella and Clostridium perfringens vaccine and objective and develop a more detailed understanding of glycobiosynthetic pathways in Coxiella and other pathogenic bacteria. Large scale virenose synthesis The robust and reproducible synthetic route to access 6-deoxy-3C-methyl-D-gulose (D-virenose) has been established and optimised. Briefly, the chemical synthesis starts from low-cost D-galactose that is initially locked into a furanose form. Further application of protecting groups allows selective reduction of the hydroxyl group on C6 and installation of methyl group on C3. A final deprotection gives desired D-virenose building block in a respectable overall yield. The process has been optimized for large scale preparation and is now possible to process >100g per step. Glycosylation and Glycoconjugation The glycosylation reaction between suitable donor/acceptor allowed the preparation of Vir1-4Vir with both a or ß configuration. Further glycosylation of each disaccharide allowed the installation of tether that was successfully used to immobilize each disaccharide on a protein scaffold. Sugar nucleotide synthesis Currently, we have designed and fully executed the chemical synthesis of both a- and ß- protected D-virenose phosphate. An a-D-virenose phosphate obtained through the phosphorylation reaction with tetrabenzyl pyrophosphate and a ß-D-virenose phosphate was synthesised using Mitsunobu phosphorylation with dibenzyl phosphate. The full de-protection and chemical coupling of nucleotide is currently underway. The obtained disaccharides will be essential as standards to identify the native structure and linkage of the LPS while the sugar nucleotide will provide substrates to identify the enzyme involved in the LPS biosynthetic pathway. All those will be readily accessible with the large-scale route established. Biochemical assignment of Coxiella O-antigen pathways We have expressed and purified the thirteen most likely proteins to be involved in Coxiella O-antigen sugar nucleotide biosynthesis, and also expressed all of the putative glycosyltransferases from Coxiella. Our approach to characterising these proteins is to prepare controls from other organisms that perform similar reactions to those required for D-virenose biosynthesis. We have completed the development of the necessary control reactions, and are now identifying enzymes from their biochemical properties. Two key enzymes have been positively identified, and we are completing the characterisation of these. We expect to characterise further enzymes at a quickening pace now that we have the first successes and all the controls in place. Clostridium perfringens carrier proteins for dual vaccine This area of research continues as in the original proposal. As outlined above we have cloned and added PglB recognition sites (glycotags) to the C. perfringens protein NetB. In addition, we have effectively de-toxified the epsilon toxin (EtxA) vaccine candidate, but this construct demonstrates lower toxin neutralising antibodies. We are currently constructing an optimised EtxA construct that is toxin neutral, but produces sufficient antibodies to elicit protection from C. perfingens infection in ruminants. At the LSHTM constructs are being generated to optimise EtxA as an acceptor protein for glycosylation with the Coxiella O-antigen, once elucidated. PglB recognition sites have been added to EtxA, and has been glycosylated with the O-antigen from Francisella tularensis using the PGCT system. Additional Coxiella studies The Wren and Titball groups have had a long-standing collaboration with Phil Felgner (University of California, Irvine), who is a world leading expert in the use of protein arrays to identify subunit protein vaccines from a variety of bacterial pathogens. One such study has identified an excellent protein candidate from Coxiella burnetti. In other independent research the Wren group have initiated work on a Brucella melitensis vaccine, coupling the Yersinia enterocolitica O-antigen (identical to that of Brucella) to the Brucella antigen Omp25. This O-antigen is compatible with the C. jejuni coupling enzyme CjPglB and initial tests have shown glycosylation of Omp25 with the Y. enterocolitica O-antigen in vivo. Through this research we have reached phase 2 of the GALVMed (AgResults) Brucella vaccine competition (https://brucellosisvaccine.org/). Objective (iii) Improve the utility, efficacy and applicability of Protein Glycan Coupling Technology for glycoconjugate vaccinology and for further glycobiotechnological applications. 2.6.1 Expanding the specificity of CjPglB for glycoengineering applications - We have several advances currently ongoing at the LSHTM. These include: (i) the generation of the N311V, S80R, Q287P mutations in C. jejuni PglB that has been shown to enable galactose transfer, (ii) the characterisation of C. sputorum PglB, the first PglB orthologue found to have a different sugar substrate specificity to the enzyme from C. jejuni (patent PCT/GB2018/050647), (iii) the utilisation of PglL from Neisseria meningitidis and Acinetobacter baumanii and characterisation of the proposed recognition sequence WPGNNTSAGV. 2.6.2 Chromosomal expression of PglB - This methodology has been fully validated in our current studies. We have used Mobile Assisted Genome Integration on Chromosomes (MAGIC) technology to introduce the PglB locus (patent US14/655,128 and EP14705855.6, MAGIC technology). In addition, we have identified optimal constitutive promoter strengths to enable the assembly of glycoconjugates without the addition of an inducer compound in the culture medium. 2.6.3 Glycofishing - This objective has yet to be tested in Coxiella due to difficulties working with the wildtype strain at DSTL. The methodology has been validated in other bacteria (eg Citrobacter and Vibrio species). We have developed an in vivo assay to characterise any potential WecA and are currently testing the specificity of the assay. 2.6.4 Glycotagging - The ability to add further N-linked glycosylation sites to a given protein (glycotag) has been fully validated in this project is now routine (eg the introduction of a deca-glycotag into test carrier proteins ExoA).
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
 
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 04/2019 
End 04/2021
 
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 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, 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 01/2019 
End 12/2020
 
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 Sub-award from BBSRC Impact Acceleration Account (BB/S506722/1). Dr Adam Balic 'Dendritic cell targeting to enhance vaccination efficacy in immunologically naïve chicks'
Amount £9,544 (GBP)
Funding ID BB/S506722/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Testing poultry vaccines
Amount £54,000 (GBP)
Organisation Bioproperties Ltd 
Start 02/2019 
End 02/2020
 
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 Academic/University
Country United Kingdom
Start 10/2018 
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 Universal protection against Streptococcus pnuemoniae
Amount £989,000 (GBP)
Funding ID MR/R001871/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2018 
End 04/2021
 
Description Glycan synthesis 
Organisation Iceni Diagnostics
PI Contribution Exchange of ideas and staff with regards the chemical synthesis of glycans and sugar phosphates.
Collaborator Contribution Exchange of ideas and staff
Impact Improved methods for the chemical synthesis of glycans and sugar phosphates.
Start Year 2017
 
Description Zoetis Development of poultry vaccines 
Organisation Zoetis
Country United States 
Sector Private 
PI Contribution Technology to make cheaper more effective vaccines
Collaborator Contribution Testing of vaccines in poultry by Zoetis
Impact Early stage as vaccines are being prepared
Start Year 2017
 
Title Attenuated Streptococcus suis bacterial cells 
Description Novel low cost Streptococcal suis vaccine 
IP Reference GB1704101.2 
Protection Patent granted
Year Protection Granted 2017
Licensed Commercial In Confidence
Impact Improved pig vaccine
 
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 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 Whole Cell Vaccines 
Description Development of Protein Glycan Coupling Technology for vaccines for veterinary purposes 
IP Reference GB1603958.8 
Protection Patent application published
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact The establishment of vaccine technology platform to engineer low cost recombinant glycoconjugate vaccines
 
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 BBC Breakfast 
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 Expert opinion of an outbreak of E. coli
Year(s) Of Engagement Activity 2018
 
Description BBC Breakfast 
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 Interview on Antibiotic resistance, Vaccine development and Government plans
Year(s) Of Engagement Activity 2019
 
Description BBC News 
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 Interview on Antibiotic resistance, Vaccine development and Government plans
Year(s) Of Engagement Activity 2019
 
Description BBC News at 10 
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 Explained consequence of an E. coli outbreak
Year(s) Of Engagement Activity 2018
 
Description Feature for BBC2 Food Detectives programme 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Professor Stevens took part in a BBC2 Food Detectives programme which featured Campylobacter in chickens. He helped to design a survey of Campylobacter contamination in a sample of fresh chicken obtained from Scottish retailers and interpret the findings. This were close to Food Standards Agency findings, which were broadcast as part of an interview with Prof. Alice Roberts. The interview took place at The Roslin Institute and included a lay description of Campylobacter, the importance of chickens as a reservoir of infection and strategies by which consumers can protect themselves. It also highlighted BBSRC-funded work at Roslin to mitigate the problem based on genetic selection (this project) and vaccines (other BBSRC projects held by Prof. Stevens).
Year(s) Of Engagement Activity 2016
URL http://www.bbc.co.uk/programmes/b077rgd4
 
Description Multiple school visits and open days 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Multiple visits to state schools under the Speakers for Schools scheme giving talks to groups of 50-100 pupils. Open days at Imperial College involving over a hundred students, teachers and parents.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
 
Description Outreach event at local scout group 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Delivered an interactive workshop to a local scout group (15 attendees). This sparked many questions from the children attending on the day, and excited the leaders. The leaders reported that the group retained interest and excitement in science after the event.
Resources were developed for use with other groups in the future.
Year(s) Of Engagement Activity 2019
 
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 Public lecture entitled Confronting the Microbial Menace in Our Food'. Professor Mark Stevens 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The inaugural lecture of Professor Mark Stevens was held on 30 October 2017 on his BBSRC-funded research to identify bacterial and host factors influencing the ability of Salmonella, Campylobacter and E. coli to colonise farm animals and cause disease. It was attended by children from local schools, members of the public, students at The Roslin Institute and wider University of Edinburgh and posted online.
Year(s) Of Engagement Activity 2017,2018
URL https://media.ed.ac.uk/media/Inaugural+lectureA+Confronting+the+microbial+menace+in+our+food/1_x5k5e...
 
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/