A novel platform for producing cell-free third generation Marek's disease virus vaccine

Marek's disease virus (MDV) infects chickens and causes a deadly lymphoproliferative disease which is only controlled by mass vaccination. The most effective vaccine against MDV is an attenuated MDV (weaken form of virus) which infects chickens but does not cause the disease. In chickens, both MDV and the "gold standard" MDV vaccine remain within the infected cells (cell-associated) and cell-free form of virus (a virus which can stay infective outside of cell) is only produced by a specific cell type located in the feather follicles. Cell-free virus is shed from the feathers of infected or vaccinated birds and these cell-free viruses infect other birds after inhalation of the infected dust in the poultry farms. Currently, 22 billion doses of MDV vaccine are administered to chickens annually. Poultry vaccine manufacturers currently produce MDV vaccines in cells isolated from fertilized and embryonated eggs. Therefore, the cost of vaccination is very high as the vaccine has to be stored and transported in very low temperatures (liquid nitrogen) which requires expensive equipment, trained individuals, and special handling procedures. Another disadvantage of the current vaccine is that any error in storage and handling can reduce the effectiveness of the vaccine. We aim to produce a vaccine that is cell-free and thus it does not require very low temperatures for storage or transportation. In this project, we will develop a platform for the production of cell-free MDV vaccine by generating cells from feather follicles with the capability to produce the virus in a cell-free form. We will establish methods to increase the capacity of the cell lines to produce the cell-free MDV vaccine in the laboratory, and finally we will examine the immunogenicity and efficacy of the MDV cell-free vaccine in chickens.

Both pathogenic serotype 1 Marek's disease virus (MDV1) and vaccine strain, CVI988-Rispens, are highly cell-associated viruses. One of the major issues with the current MDV vaccination strategy is the associated cost of vaccinations owing to the cell-associated nature of the vaccine strain of virus which requires cold chain for storage and transportation. In vivo, feather follicle epithelial cells are the only known cells capable of producing cell-free MDV viruses which are heat resistant and can stay infective in the environment. We have developed methods to isolate feather follicle stem cells (FFSCs) which can potentially divide indefinitely, support MDV replication and generate cell-free MDV virus in vitro. Here, we will establish methods to increase the yield of cell-free MDV vaccine from FFSCs cell lines and examine temperature stability of the cell-free MDV vaccine in vitro. To achieve this goal, we will determine the best combinations of methods developed for preparation of high titre human alphaherpesviruses. Moreover, we will examine whether induction of cell differentiation can increase cell-free virus yield in vitro. Finally, we will determine the immunogenicity and efficacy of the cell-free MDV vaccine in a challenge experiment in the absence or presence of maternal antibodies in vivo.

This is an Industrial Partnership Awards (IPAs) application in which Zoetis, our industry partner, will contribute in cash equivalent to 10% of the full project costs. The aim of this proposal is to develop a platform for generation of cell-free form of vaccine strain of Marek's disease virus serotype 1 (MDV-1), examine temperature sensitivity of cell-free vaccine and determine the immunogenicity and efficacy of the vaccine to control Marek's disease (MD) in chickens. Although the impact of this research will initially benefit the academic community, the results from this study will have long-term impact on vaccine development, and may lead to the development of cell-free vaccine against MD. The development of cell-free MDV vaccine can reduce the cost of vaccination against MD as it can remove the cost associated with storage and transportation of cell-associated vaccine, and this will increase the availability of MDV1 vaccine to backyard poultry farmers in low and middle income countries. Poultry and poultry products are the cheapest and most accessible source of animal protein and demands for these products have increased exponentially. Compared to the other livestock sectors, the modern poultry production methods have the most efficient feed-to-meat conversion ratios with lowest global warming potential. It is estimated that poultry will account for 46% of meat consumed by 2022 around the world. Poultry meat is important to the UK, not just because of the food it provides but because it contributes £3.3 billion to UK GDP, with every £1 billion generating another £1.3 billion in the rest of UK economy. The industry supports 35,400 direct and a further 37,900 indirect jobs in the wider supply chain (Oxford Economics, 2013). Infectious diseases are a continuous threat to poultry industry through losses or reduction in production and animal welfare. MD is one of the major diseases of poultry which causes serious economic losses and the global estimate of losses from MD is approximately US $2 billion annually. The outcome of this research will provide crucial information which could be used to generate more effective vaccines providing protections against infection in partnership with poultry vaccine industry such as Zoetis. Although the focus in this proposal is on MD, our results will have a wider impact by providing underpinning knowledge for similar studies with other economically important avian pathogens, as MDV strains are used as a vector for generation of immunity against other infectious diseases. The results of the project will be communicated to The Pirbright Institute stakeholders, such as BBSRC and DEFRA. The reagents and scientific knowledge generated will be published in peer-reviewed journals, and in presentations at meetings for scientists, and the impact of the project will also be publicized by communication with the farming community and the public through our website, workshops, training courses and exhibitions. The Pirbright Institute is committed to the development of highly skilled researchers and the postdoctoral scientists who will be recruited for this post will receive excellent training in vaccine development, cell culture techniques, confocal microscopy, flow cytometry, cellular and molecular techniques, thereby contributing to the wider training, innovation, skills and capability of the UK science with consequent boosting to science based industries and the UK economy.