Recombinant vaccines for poultry: the use of attenuated Eimeria acervulina as a novel live delivery vehicle for viral vaccines

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

Abstract

More than 50 billion chickens are produced each year in the world and control of infectious diseases is of primary concern to the poultry industry. Viral, bacterial and parasitic infections compromise the economics and efficiency of meat and egg production, cause severe animal welfare problems and in some cases have serious potential for infecting humans. Demand for effective, cheap vaccines against a broad panel of pathogens has never been higher, particularly set against the pressing needs of global food security. For many viral infections of poultry, we understand which parts of the virus are needed to induce strong protective immunity and could be in a good position to develop new kinds of recombinant 'vectored' vaccines that would be safe, cheap and effective. However the development of organisms that could be used as the vaccine delivery 'vectors' has so far seen commercialisation of only two based on fowlpox virus (FWPV) and herpes virus of turkeys (HVT). Both of these suffer some drawbacks, especially because they have to be given by individual scarification or injection and cannot be administered in a simpler way such as in drinking water or by aerosol spray over large numbers of chicks. Eimeria are host-specific protozoan parasites that transmit between birds by the faecal-oral route and rapidly establish acute intestinal infections that are self-limiting (7-14 days) and induce life-long protective immunity. In poultry they cause coccidiosis, one of the most globally important infections that afflicts the industry, causing severe losses unless controlled by chemotherapy or vaccination. Vaccines using laboratory-derived lines of live-attenuated Eimeria are given in the drinking water and are well established and very effective. The world-leading Eimeria vaccine is Paracox, manufactured by Intervet/Schering-Plough Animal Health, and this sells > 1 billion doses each year and has an exemplary record of safety and efficacy. In our lab we have recently developed methods that allow us to manipulate the genetic material of Eimeria parasites which means we can now make stable transgenic parasite lines; this means it is possible to try and develop Eimeria parasites as vaccine vectors for vaccinal antigens. Recombinant vaccines based on an Eimeria vector would have several advantages over existing vaccines. Because the parasites have large genomes it is likely that we will be able to insert and express vaccine antigens from several different organisms within a single parasite line so a single vaccine could protect against several diseases. These parasites are already licensed as live vaccines and are known to be extremely safe; they infect only chickens and they are completely cleared from the host by 14 days after vaccination. They are known to induce a broad range of potent immune responses so they are likely to be useful for inducing immunity to different kinds of pathogens (viruses, other parasites, bacteria). We propose to investigate the vaccine vector potential of an attenuated Eimeria parasite (E. acervulina HP, one of the Paracox parasites) using well characterised antigens from two important viruses of poultry. The first is infectious bursal disease virus (IBDV), which causes Gumboro, an immunosuppressive disease of poultry that poses severe problems in health and productivity and also interferes with the efficacy of vaccination programmes. IBDV is a particular problem in the broiler industry at the moment with new virulent and variant strains emerging that are able to break through existing vaccines. The second is infectious laryngotracheitis virus, a persistent problem particularly in breeding and commercial layer flocks and for which strong cellular immunity as well as antibody responses are required for effective control.

Technical Summary

Chickens are the most numerous livestock species in the world. Industrial and political demand for effective, cheap vaccines against infectious diseases that can compromise poultry production, cause severe animal welfare problems and have serious potential for infecting humans has never been higher, particularly set against the pressing needs of global food security. Whilst immunoprotective antigens have been identified for many viral pathogens that infect poultry the commercial development of delivery vectors has been limited to fowlpox virus and a herpes virus of turkeys, both of which require invasive single bird treatment. The ability of Eimeria to induce potent life-long protective immunity after a single self-limiting, host-specific infection and the robust longevity of the oocyst phase of the lifecycle, combined with the recent development of stable transfection strategies for these parasites, promotes their use as novel vectors. Attenuated Eimeria parasites are already extensively used as anticoccidial vaccines. In partnership with Intervet/Schering-Plough Animal Health we propose to investigate the vector potential of Eimeria using Eimeria acervulina as a model to deliver well characterised antigens from two persistently problematic viruses of poultry, infectious bursal disease virus (IBDV) and infectious laryngotracheitis virus (ILTV). Transfection constructs developed for use with Eimeria tenella will be tested with E. acervulina and modified to express the vaccinal antigens IBDV VP2 or ILTV gB, supported by new next-generation derived genomic resources for the recipient. Constructs designed to control the site or timing of transgene expression will be developed and tested to identify the best combination on the basis of ability to induce host immune responses and provide protection against viral challenge. This project is expected to produce parasite lines suitable for use as new vaccines and data supporting the use of Eimeria as vaccine vectors.

Publications

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Chapman HD (2013) A selective review of advances in coccidiosis research. in Advances in parasitology

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Clark EL (2017) Are Eimeria Genetically Diverse, and Does It Matter? in Trends in parasitology

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Pastor-Fernández I (2018) Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens. in International journal for parasitology

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Blake DP (2015) Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development. in Proceedings of the National Academy of Sciences of the United States of America

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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 We generated a series of populations of transgenic Eimeria tenella parasites that express three vaccine antigens derived from two viral pathogens of poultry (infectious bursal disease virus, IBDV; infectious laryngotracheitis virus, ILTV). For each of these targets we generated two populations which express a soluble version of the antigen, expressed at either a high or low level (relative to endogenous E. tenella antigens; data derived from RNAseq libraries) and two further populations that express secreted or GPI-anchored versions of the antigen, expressed at a high level. The methodology relies on the assembly of Eimeria-specific expression cassettes within a plasmid vector that co-expresses either mCitrine or mCherry fluorescent reporters also under control of an Eimeria-specific promoter. Individual plasmids were used to generate the transgenic parasite populations by nucleofection of the sporozoite stage in vitro using a restriction-enzyme mediated integration enhancement to promote high copy number integration at random sites. After passage through chickens, with enrichment for transgenic parasites by FACs sorting of offspring oocysts, populations were then sent to our industrial partner for testing immunogenicity and efficacy in vivo.

Characterisation of the transgenic parasites and analysis of the potential of Eimeria parasites as novel vaccine vectors is now completed. Antigens were successfully expressed and we were able to demonstrate a low level of antibody responsiveness to viral antigens expressed as soluble, secreted or surface-tethered proteins within the parasites.
Exploitation Route Applications are in the field of vaccinology and the potential use of a host-specific gut parasite for delivery of antigens to the chicken. Because of the work we have done in this grant, we were successful in securing funding to continue development of the vaccine vector through two H2020 consortium grants. The outcomes from these grants have built on the findings from the BBSRC grant, resulting in further publication. In addition we were successful in winning funding from the internal RVC concept development fund and through this mechanism are continuing to develop the Eimeria vector technology, specifically towards the generation of multivalent vaccines against multiple species of coccidiosis.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description Findings from the project were used in two consortium grants funded by EU H2020. This led to additional development of the technology with new publications and additional internal funding from the RVC Concept Development Fund. This latter funding is to take forward the Eimeria vector technology for development and testing of multivalent transgenic populations of Eimeria tenella as improved vaccines against coccidioisis in chickens. The funding supports a PDRA for 15 months and also supports the use of an external consultant who is working with us to identify and prioritise industry partners for taking the concept forward. This phase of the work will continue until Autumn 2019. It is at an early 'emerging' stage in terms of impact.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Houghton Trust Small Grant Award (2016/2017)
Amount £8,000 (GBP)
Organisation The Houghton Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 10/2017
 
Description Response mode highlight 'Vaccine'
Amount £400,104 (GBP)
Funding ID BB/P003931/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2016 
End 12/2019
 
Description Tackling Losses from Terrestirial Animal Diseases: SAPHIR
Amount £595,000 (GBP)
Funding ID 633184 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 09/2015 
End 09/2018
 
Description Tackling losses from Terrestial Animal Diseases: PARAGONE
Amount £314,000 (GBP)
Funding ID 635408 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 09/2015 
End 04/2018
 
Description early post-doc mobility fellowshiop
Amount SFr. 120,000 (CHF)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 03/2014 
End 09/2015
 
Title Improvement to transfection technology for Eimeria species 
Description Adapted methods for nucleofection of eimeria sporozoites to use smaller numbers of parasites, and be able to select transgenic parasite populations rapidly by FACs analysis of output oocysts. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Allows for more rapid screening of candidate vaccines (expressed in the Eimeria vector platform) in both in vitro (cell culture) and in vivo (chickens). This improved methodology is now being exploited in two H2020 projects, funded within our lab. Publication on the methodology is in preparation. 
 
Title Laboratory protocols for Eimeria 
Description Protocols specific for the passage, purification and manipulation of Eimeria parasites were introduced to TANUVAS, Chennai and IVRI, Bareilly. Development and validation of methods for this project at TANUVAS, IVRI and RVC yielded a comprehensive protocol describing sample collection, preparation and diagnostic analysis for use with EImeria (for the first time). Protocols specific to the development of attenuated vaccines using local parasite strains and to culture of E. tenella in embryonic chicks and cell culture were provided to TANUVAS in support of parallel projects. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Support and expansion of parasitology research at TANUVAS and IVRI; provision of robust sampling methods for parasite surveillance activities; publication of several papers in local and international journals