Selection Versus Mutation: Reducing the Risk of Vaccine Reversion

Vaccination against numerous endemic pathogens is an essential component of the poultry industry. Without these vaccines chickens would succumb to infection at an early age reducing the productivity of the industry well below sustainable levels. IBV is an endemic virus that causes severe disease outbreaks in chickens worldwide. Effective and economically viable vaccines against IBV are available and mainly produced from pathogenic virus strains by passing in eggs approximately one hundred times. During these passages the virus accumulates multiple sequence variations from the original pathogenic sequence. This ultimately leads to attenuation of the virus and the production of a live attenuated vaccine. These vaccines have lost their ability to cause disease but still elicit a protective immune response in the chicken, thus protecting the bird from future infections. However, as these are live viruses the potential for to revert back to a pathogenic form is considerable considering the few sequence changes between wild and vaccine strains.

Despite the importance of these vaccines to the poultry industry and the risk of reversion, the processes that occur and the selective forces that drive virus attenuation during egg passage are unknown. Importantly, the differential contribution of virus sequence mutation compared to the selection of minor variants already present in the virus population has not been determined. Understanding these basic processes is essential to the development of future vaccines to reduce the threat of reversion.

This study will use passaged pathogenic IBV strains produced in the same way as vaccines. In parallel we will use a unique system that allows us to passage a single virus clone rather than a mixed virus population. Using contemporary deep sequencing technology we will study the molecular changes that occur at fine resolution during the attenuation process. This will for the first time reveal how a mixed population of virus changes during vaccine manufacture and the extent to which individual viruses can mutate. These results will then inform a series of studies that manipulates the forces that drive virus change. The first will use IBV strains that contain a protein from another strain that influences the immune response in the chicken, and the second will use viruses that mutate much faster than wild type viruses. By passaging and deep sequencing these viruses in the same way as the wild type viruses, we will understand how different forces drive virus sequence mutation. These recombinant passaged viruses will then be tested to determine if this process has led to attenuation and also if they maintain the potential to infect other chickens that are exposed to vaccinated birds.

Ultimately this research will reveal how IBV is attenuated by egg passage and identify key regions of the genome that prevent the virus from causing disease but do not impair its potential as a vaccine. Moreover, we will further develop our understanding of how different pressures influence the attenuation process and potentially identify ways to improve the process of vaccine design by engineering attenuated viruses. By understanding and manipulating the processes that govern virus attenuation and vaccine production we aim to identify ways of reducing the danger of vaccine strains reverting and causing damaging disease outbreaks.

Avian coronavirus Infectious bronchitis virus (IBV) is responsible for an important disease affecting the global poultry industry; a potential threat to food security. Live attenuated vaccines are crucial for the protection of large commercial poultry flocks. IBV live attenuated vaccines are produced by multiple passage of a virulent virus in embryonated hens' eggs, an empirical process believed to result in spontaneous mutations resulting in attenuation. Viruses attenuated by this approach may only have a few mutations that are responsible for loss of virulence or the variation in mutations results in vaccines with differing effectiveness. A major drawback of the method is back-mutation and re-emergence of virulent virus. Recent work has shown that the number of mutations identified following attenuation results in a low number of amino acid substitutions, <20, in the whole genome. Other recent work has shown that coronaviruses, in contrast to other positive sense RNA viruses, encode a proof-reading activity as part of their replicase repertoire. This raises the possibility that attenuation of IBV may result from selection of minor variants present in the initial inoculum rather than the accumulation of spontaneous mutations.
This project presents a unique opportunity by combining two fundamental technologies, second generation deep sequencing and IBV reverse genetics, to understand, for the first time, the process of attenuation used to generate IBV vaccines. Knowledge gained will significantly help to reduce the possibility of reversion. Moreover, by decreasing the proofreading ability of IBV it may be possible to develop vaccine strains that elicit a protective immune response but are short lived as they will accrue too many mutations to survive in the environment. Reducing the chances of reversion and the danger that live attenuated IBV vaccines may be responsible for the emergence of new pathogenic variants as a result of recombination with field isolates.

Poultry is an important food source worldwide, there are an estimated 55 billion chickens including 5 billion for egg production. Viral diseases are a constant threat to the poultry industry through loss or reduction in the production, decreases in egg production and quality and affects on animal welfare. IBV causes an acute highly contagious and economically important respiratory disease responsible for economic losses to poultry industries across the globe and is a threat to food security. IBV also affects both the production and quality of eggs and despite the availability of live and inactivated vaccines continues to be a major problem worldwide. Live attenuated vaccines are produced by multiple passages of virulent isolates in embryonated domestic fowl eggs. This procedure is empirical, the mechanism of attenuation is unknown, often a fine balance between attenuation and loss of immunogenicity and has the major drawback of reversion to virulence.
The ultimate aim of this proposal is to determine how multiple passages of the virus results in attenuation and to use the information gained for the development of safer IBV vaccines by reducing their ability to revert to virulence if they escape into the environment. There are several beneficiaries of the work planned in this project.

BBSRC: Food security is recognised as an important research priority in the BBSRC 2010-2015 Strategic Plan. Results from this proposal will provide crucial information on how vaccines used to control an important avian endemic pathogen are attenuated, and information for the development of safer and more efficient vaccines ensuring that poultry farming remains not only a secure food source but also increases the economic competitiveness of the UK.

Poultry industry: IBV is a major challenge both to the UK and global poultry industry. In the UK, poultry production in 2010 was 904 million broilers and 34.5 million layers producing 9.7 billion eggs. The UK poultry industry is more than 90% self sufficient and in 2010 contributed around £4 billion to the UK economy supporting 60,000 associated jobs. Improved efficiency of the industry, through continued protection against endemic diseases such as IBV and the development of more efficient and safer vaccines, particularly against new and continually emerging variants of IBV, will have positive knock-on benefits both socially and for the UK economy. A Defra-funded report estimated that IBV affects 22 million birds in the UK incurring an overall cost of £23 million. At present, the mechanistic events resulting in attenuation is poorly understood; research to understand the molecular basis for attenuation, as described in this project, will provide the information to produce more effective and safer vaccines. This will result in improvements in animal welfare, reduced losses to the poultry industry and to reduce risks to food security.

Vaccine companies: The IAH coronavirus group has established collaborations, including direct support, with several vaccines companies that have resulted in ongoing assessment of potential vaccine candidates. This project is supported by one of the top poultry vaccine companies, Pfizer Animal Health, by the provision of virus passages used to produce a vaccine against the newly emerged IBV QX variant. The data generated during this project will allow us to understand how IBV is attenuated for the generation of vaccines and provide information for the design of safer vaccines.

Academia and Training: Results with respect to how viruses diverge and evolve on serial passage, especially in the absence of proof reading with respect to coronaviruses will be of interest to the scientific community and will be published in peer reviewed journals and presented at national and international scientific meetings. The project will provide professional training to a post-doctoral scientist, especially relating to virology and bioinformatics in respect to deep sequence analysis.