Investigating Host and Viral Factors for Improved Design of Future Live Attenuated Vaccines for IBV

Vaccination against endemic pathogens is an essential component of the poultry industry, without which chickens would become infected at an early age. This would reduce productivity and push food security beyond sustainable levels. Infectious bronchitis virus (IBV) is a coronavirus that causes respiratory disease in chickens, making them more susceptible to bacterial infections. After infection, they achieve lower weights and produce fewer high-quality eggs. It has been estimated that a 10% reduction in IBV incidence globally would be worth £654million to the poultry industry.
An effective vaccine to IBV is therefore critical for both welfare and economic reasons. The best vaccine strategies for IBV are "live attenuated" types, meaning a weakened form of the disease-causing virus is used to vaccinate chickens, generating good immunity. These vaccines are produced by growing disease-causing (pathogenic) viruses in eggs up to one hundred times, during which multiple genome changes can occur. Our understanding of how this process works is limited. Whilst these vaccines have lost their ability to cause symptoms, they still retain the ability to induce protective immune responses in chickens, thereby protecting them from disease. However, some vaccine strains have been reported to evolve, causing outbreaks of disease in flocks after vaccination. This occurs as vaccine virus genomes change and there is a risk, they may regain their pathogenic capability (reversion), producing disease in vaccinated birds i.e. the vaccine virus causes new outbreaks. Understanding better how both vaccine viruses are weakened and the host pressures after vaccination drive reversion are key to designing more stable vaccines.
In a previous project (BB/L003988/1) 2 IBV viruses were weakened (a commercial vaccine and a lab-adapted strain) by passaging in eggs to identify how the viruses changed. We deep sequenced these viruses every 10 passages to understand how they change. We know from existing datasets that some changes are shared between both weakened and pathogenic viruses. We also know that some changes are exclusive to each type. Moreover, sequence changes are influenced by factors including restricted diversity and dilution factors during egg passaging. We do not know how these changes will impact reversion after vaccination.
The overall aim of the project is to produce a detailed profile of the viral dynamics-host interactions of IBV vaccination in chickens to gain major insights into the virus biology and host responses. This will answer 3 research questions:
1. Are there changes in the virus during this weakening process that can be used to improve future vaccines?
2. How do vaccine virus's genomes changes after vaccination into chickens and do these changes make them more likely to cause reversion?
3. What host genes are expressed, and do they help drive vaccine viruses to evolve in chickens?
We will use deep learning will identify genomic patterns in viral genomes during the weakening process. We will characterise how vaccine viruses change after vaccination into chickens, comparing changes to those in pathogenic viruses, to measure the likelihood of reversion. We will characterise cellular gene expression, after vaccination and use machine learning to identify cellular responses driving changes in the vaccine virus genomes. Finally, we will combine datasets and use machine learning to make predictions on which sequence changes are important in the processes of weakening viruses, and reversion. These predictions will be re-inserted back into a virus and their impact on chicken cells measured.
This research will identify genome changes involved in the occurrence and likelihood of reversion and reveal how they will change post-vaccination. These results will further our understanding of processes impacting virus genomes both during attenuation and after vaccination, that can be used in future next generation vaccines.

Infectious Bronchitis is the most economically important infectious disease affecting poultry globally. We have shown previously that attenuation of the avian coronavirus, infectious bronchitis virus (IBV) by serial egg passaging is likely a multifactorial process rather than being driven by a single pathway. Little is known regarding the mechanisms underpinning this process, with few studies having evaluated vaccine virus genome sequence stability post-vaccination and its contribution to reversion to virulence, that can seed new outbreaks driven by vaccine viruses.

The following research questions will be considered:
1. Are there changes in the virus during the attenuation process that can be used to improve future vaccines?
2. How does the genome sequence stability of vaccine viruses change after vaccination, and do those changes contribute to reversion of virulence?
3. What role do host genes have in driving vaccine virus evolution in vivo?

We will use deep learning methods to identify patterns within 52 existing whole genome sequence datasets generated during egg passaging of 2 viruses (QX and M41-CK) to attenuation. We will then generate novel sequence information for whole S gene/whole IBV genome to explore virus genome sequence changes occurring post-vaccination. Changes will be compared with pathogenic IBV isolates to link to phenotype. We will use spatial transcriptomics to identify changes in host gene expression within tissues from vaccinated birds. This will identify cellular responses driving virus changes at a cellular level. We will take the outputs of these three components, combining them into a single dataset and process them using deep learning to make final predictions of genomic markers or signatures contributing to attenuation and validate these ex-vivo.

This work will identify better ways to attenuate viruses for use as live attenuated vaccines, whilst minimising the potential for reversion to virulence.