VACCINE. Development of a novel yeast-based oral subunit vaccine against Eimeria spp. in chickens

Lead Research Organisation: Royal Veterinary College
Department Name: Pathology and Pathogen Biology

Abstract

Yeast species such as Saccharomyces cerevisiae are useful tools for the high yield production of recombinant proteins and have a Generally Regarded As Safe (GRAS) status. They are capable of performing several complex post-translational modifications that are not achieved in many other expression systems, and are easily grown to very high densities producing large quantities of stable particles. Recently, the idea of using S. cerevisiae as a delivery vehicle for cancer, viral, and bacterial vaccines has been explored, inducing robust humoral and cellular immune responses. In addition to using yeast to produce a vaccine antigen of interest, the yeast cell itself has been shown to have adjuvant-like properties and has the potential to activate both inflammatory and phagocytic receptors expressed on antigen-presenting cells. Our preliminary data demonstrates that freeze-drying recombinant yeast cultures expressing viral protein at their surface renders the recombinant yeast completely non-viable (unpublished). However, the freeze-drying process does not alter conformation of these proteins, as surface expression is equal in live and freeze-dried yeast as quantified by flow cytometry and Western blotting. This has interesting implications in vaccine design as a non-viable S. cerevisiae is not categorised as a genetically modified organism (GMO) and such a killed vaccine would not be subject to GMO regulations. Additionally there would be no need for refrigeration of the freeze-dried yeast, reducing transport and storage costs. Vaccines based upon S. cerevisiae are likely to be particularly valuable against diseases of farmed poultry, where safety, scalability, stability, delivery and cost are crucial. In one example modern poultry production relies on effective control of Eimeria, but current approaches using drugs and live vaccines require improvement. In recent years a panel of coccidial antigens have been identified as vaccine candidates, each individually capable of inducing up to a 65% reduction in oocyst output. Now, the focus is shifting from antigen discovery to antigen formulation and delivery with yeast being a leading option, especially given the importance of T-cell mediated responses in anticoccidial immunity.

Technical Summary

In the work proposed here we will develop Saccharomyces cerevisiae as an oral vaccine vector platform for use with poultry, employing freeze-drying to inactivate and stabilise vaccine formulations. Non-viable yeast cells are not considered as GMOs (EU directive 2001/18/EC) and benefit from Generally Regarded As Safe (GRAS) status. We will build on proof of concept studies with pigs to validate the freeze-drying process, comparing immunity induced in chickens after vaccination with live yeast cultures, bacterial expressed recombinant protein and natural live parasite exposure. Development of a non-GMO vector system with no requirement for a cold chain and which can be included in animal feed would revolutionise poultry vaccination.

The yeast system will be developed using three well-established anticoccidial vaccine candidates. Coccidiosis caused by Eimeria parasites remains a major animal health and welfare concern with significant impact on food security and economic productivity. The proteins apical membrane antigen 1 (AMA1), immune mapped protein 1 (IMP1) and microneme protein 3 (MIC3) all show robust anticoccidial vaccine potential against Eimeria tenella, one of the most important species of the genus. To date, these antigens have only been tested individually. Here, we will investigate multiple combinations of these antigens to identify an optimal multi-valent vaccine which can be used in the rational development of equivalent vaccines against other high impact Eimeria species.

Major outputs will include understanding of the breadth of immune responses, systemic and local, stimulated by oral yeast-vectored vaccination in different chicken body compartments, indicating the range of pathogens which may be rationally targeted in future applications. Finally, we will use specialised facilities to test in vivo protection stimulated by the optimised vaccine under simulated field conditions, beginning to bridge the gap between tool development and practical use

Planned Impact

The work proposed has direct relevance to the Cross-Councils highlight 'novel tools and technologies for vaccinology'. The project falls within BBSRC strategic research priority areas: (1) Animal health (developing strategies to combat disease) (2) Sustainably enhancing agricultural production (improving survival/longevity) and (3) Welfare of managed animals (alleviation of disease). Outputs include establishment of freeze-dried S. cerevisiae as a safe and stable oral vaccine vector for poultry, using the coccidial parasite E. tenella as an example with immediate relevance to poultry production and welfare. Outcomes will assist in increasing UK competitiveness in the global animal production market, improving animal welfare and helping to guarantee a secure supply of safe, healthy food. The following stakeholders will benefit from impact arising from this work.

1. The poultry production industry
Chicken production and welfare benefit from many vaccines, but more are urgently required. For many pathogens candidate vaccine antigens have been documented, but progress has been limited by availability of cost-effective strategies for delivery within the tight economic margins inherent to the poultry industry. Validation of freeze-dried S. cerevisiae as a safe, stable vector platform effective when delivered directly to chickens through their diet can revolutionise production, improving economic performance and welfare. Our close relationships with industry representatives can fast track the vector into field use, starting with the anticoccidial vaccine targeted here. Findings will be relevant to vaccines targeting other pathogens in chickens, as well as pigs and ruminants.

2. The animal health industry
The UK currently leads the world in the production of live attenuated anticoccidial vaccines but a major limiting factor is the requirement for production in vivo, illustrated by recent production failures and the consequential vaccine shortage. Recombinant anticoccidial vaccines have been sought for more than 30 years. The combination of effective vaccine candidates and a realistic vector system have finally brought such vaccines within reach. The scalability and stability of a yeast-based vaccine would support expansion of vaccination from the minority to the majority of chicken production, improving chicken health.

3. Animal welfare
The effective reduction of disease as a result of improved vaccine availability against coccidial and other pathogens supports the Five Freedoms implicit to animal welfare as set out by the Farm Animal Welfare Council. A recombinant anticoccidial vaccine will also reduce the requirement for chicken use in vaccine production.

4. General public and the environment
Increased efficiency in poultry production will raise poultry product availability at a lower cost for the consumer, contributing to improved food security. Consequences of improved pathogen control include a reduction in the requirement for chemoprophylaxis, reducing drug consumption, the risk of contamination to the food chain and the environment, and selection for drug resistance.
All three investigators are actively engaged in public dissemination of UK research. Students at all levels of education can benefit from the principles established in this work.

5. Skills, knowledge and training
The multidisciplinary nature of this project will provide opportunities for broad training to all staff, in addition to other members and students of each host institution, strengthening the research community in the areas of disease control and vaccine development. Broader impact can be achieved using avenues such as the UK Veterinary Vaccines Network.

6. International development
Infectious diseases impose serious costs on animal production in developing counties. Translating high quality, innovative, strategic research within UK universities into cheap, efficacious vaccines can improve economic income and alleviate poverty.

Publications

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Soutter F (2020) Poultry Coccidiosis: Design and Interpretation of Vaccine Studies. in Frontiers in veterinary science

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Blake DP (2017) Recombinant anticoccidial vaccines - a cup half full? in Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases

 
Description At the conclusion of the first year we had discovered that anticoccidial antigens expressed in yeast and delivered to chickens via oral inoculation could induce a protective immune response against low dose parasite challenge, measured as a reduction in parasite replication. We also found that the combination of multiple antigens in a vaccine provided additive protection. In the second year we have built on these results, demonstrating protection against a high dose (pathogenic) parasite challenge in terms of lesion score and body weight gain, measures of severity and economic productivity with direct relevance to industry. We have explored variation in (i) administration protocols and (ii) yeast preservation/killing approaches. We are now preparing complementary yeast lines defined by higher antigen expression and a different site of antigen localisation for use in a comparative vaccination study. The optimal yeast/antigen combinaton will then be tested in commercial-type chickens under commercial conditions.
Exploitation Route Output from this work can be taken forward by vaccine or animal feed companies to develop new anticoccidial vaccines. We also intend to prepare an application for follow-on-funding if the next trials are as promising as the last.

In the last year we have discussed the project and its results with three different prospective commercial partners under confidentiality agreements. All three expressed interest, highlighting specific questions that can be addressed in our last commercial-conditions vaccination trial as a prelude towards further discussions.
Sectors Agriculture, Food and Drink

 
Description Following our early positive results we initiated discussion with Alltech, a major animal health compnay and producer of probiotic yeasts, looking towards commercialising the work. In the second year we have also engaged with Eco Animal Health and MSD Animal Health, identifying a series of commercially relevant questions that can be answered and assist with progress towards commercial development.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Further development of anticoccidial vaccine candidates 
Organisation Proxima Concepts Limited
Country United Kingdom 
Sector Private 
PI Contribution Planning towards new anticoccidial vaccine development study
Collaborator Contribution Planning towards new anticoccidial vaccine development study
Impact Application for Innovate UK funding in preparation.
Start Year 2017
 
Title Vaccine - Immune mapped protein 1 
Description Anticoccidial vaccine candidate, identified under previous BBSRC funded project and now used in ongoing recombinant/subunit vaccine development. Data generated here used to defend the patent application. 
IP Reference EP2601211 
Protection Patent granted
Year Protection Granted 2018
Licensed No
Impact The patent is supporting a search for commercial partners to develop an associated vector system.
 
Description Emerging Eimeria species and chicken genetics in Africa, University of Kwazulu-Natal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact ~75 students and staff attended an invited plenary presentation at the University of Kwazulu-Natal, Durban, South Africa. Discussed project outcomes and follow up regarding student projects and possible project supervision. Dissemination of results to a wider community.
Year(s) Of Engagement Activity 2019
 
Description Engormix video 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Video of conference presentation 'Broilers, Eimeria and enteric microbiomes - a tale of tolerance' launched on the Engormix website, supplemented by a recorded interview.
Year(s) Of Engagement Activity 2017
URL https://en.engormix.com/MA-poultry-industry/videos/broilers-eimeria-enteric-microbiomes-tale-toleran...
 
Description Huvepharma advanced Eimeria workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Industry led training workshop - included theoretical and practical aspects in two day format.
Year(s) Of Engagement Activity 2017
 
Description Internet interview 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Interview with Engormix at the IHSIG conference, loaded onto website with international audience, advertised to network by email.
Year(s) Of Engagement Activity 2017
URL https://en.engormix.com/MA-poultry-industry/videos/damer-blake-talks-about-his-research-eimeria-geno...
 
Description Postdoc invited presentation: 12th International Coccidiosis Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact First invited presentation for a postdoctoral scientist, built network and enhanced experience/career
Year(s) Of Engagement Activity 2017
 
Description Student workshops and training, University of Kwazulu-Natal 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact ~20 students (undergraduate and postgraduate) attended a series of taught sessions and practical workshops focused on Eimeria, parasite, microbiome and chicken genetics at the University of Kwazulu-Natal, Durban, South Africa. Dissemination of results to a wider community, capacity/capability building.
Year(s) Of Engagement Activity 2019