Glycoengineering of Veterinary Vaccines

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.

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.

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-year-antimicrobial-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.