Towards control of Infectious bronchitis virus; understanding cross-protection and the genetic plasticity of IBV

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. Infectious bronchitis virus (IBV) is an endemic virus that causes severe disease outbreaks in chickens worldwide; it affects the global production of meat-type birds, due to problems in weight gain and quality, and egg production through decreasing the numbers and quality of eggs produced. Effective and economically viable vaccines against IBV are available, but multiple combinations of available vaccines are needed because the level of cross-protection against different IBV strains is insufficient. Poor cross-protection is the result of variation in a major surface protein of the virus (the spike (S) protein). New variant strains of IBV with differences in the S protein appear regularly in the field and, through analysis based on the sequence of the S protein, it is impossible to predict which vaccines will induce protection against the newly emerged viruses. Only elaborate and expensive testing in chickens elucidates which vaccine combination is needed to protect against a new strain of IBV.

This proposal will address the seemingly unpredictable nature of the virus. The availability of a unique reverse genetics system for IBV has the potential to lead to the development of a new generation of live vaccines. In this proposal we will generate recombinant viruses that are identical, except for the immunodominant S1 subunit, of the economically most important IBV strains (M41, 4/91 and QX). Vaccination-challenge experiments with the same and with different viruses will identify if there are different degrees of protection. The causes of insufficient and unpredictable levels of cross-protection are the main focus of this study. Ultimately we will determine the key regions or epitopes on the S1 subunit of the economically most important IBV strains that are responsible for inducing protective immune responses. We will use novel "epitope fingerprinting" technology to determine the key regions (epitopes) that are recognised by the antibodies induced after vaccination. Identification of key regions following vaccination with a single IBV strain or multiple strains will allow us to determine which epitopes are needed by a vaccine to induce protection. When new virus strains emerge we will then be able to predict which vaccines will be required to induce effective protection against the new virus strain.

Moreover, we will further develop our understanding of how pressure from the bird's immune responses on the virus might drive the virus to change or mutate. This will involve the passage of an IBV strain in eggs, in the same way as vaccines are produced. However, the replication of the virus will be put under immune pressure by the addition of antibodies specific for this virus. This will essentially mimic the immune pressure applied to the replicating virus, as occurs naturally after vaccination but without testing this in birds. Using contemporary deep sequencing technology we will identify the molecular changes that occur as a result of immune pressure and the process by which the virus is able to evade the applied vaccine, potentially evolving into a new variant. By understanding and manipulating the processes that govern virus adaptation after vaccination, we aim to identify ways of reducing the danger of vaccine strains changing and causing damaging disease outbreaks.
Results from this proposal will provide (1) crucial information on why vaccines used to control an important avian endemic pathogen IBV fail to induce cross-protection, (2) information for the efficient use of existing vaccines and (3) the development of more efficient vaccines, thus ensuring that poultry farming remains not only a secure food source but also increases the economic competitiveness of the UK.

The overall aim of this project is to address the unresolved question "which epitopes can induce cross-protection between infectious bronchitis virus (IBV) strains and are these epitopes prone to mutate under immune pressure?"
We will generate isogenic recombinant viruses (rIBV) expressing the immunodominant part, S1, of the S protein of the economically important IBV strains, M41, 4/91 and QX. Using in vivo vaccination-challenge experiments we will assess (i) whether homologous S1 can induce full protection, (ii) whether vaccination with heterologous S1 can induce cross-protection and (iii) if maternally derived antibodies affect the induction of protective immune responses. As the rIBVs are isogenic, any immune responses against other IBV proteins will be similar between birds. Birds will be sampled during the course of the infections to analyse the tropism of rIBV and local cellular infiltrates by immunohistology. These studies will provide a unique set of S1-specific antibodies that are induced against the different S1 sequences. These sera will be used to define the immunogenic epitopes on S1 of the 3 viruses. The epitopes will be assessed by CLIPS technology, using peptides that are fixed into defined three-dimensional structures resulting in functional mimics of complex binding sites. The epitope mapping data will determine whether the epitopes are closely located, overlapping within the S1 sequences or if they are independent and specific to the serotypes. Knowledge about the protective epitopes will be used to investigate the predictive value of S1 sequences of other IBV serotypes for protection. Finally we will test the genetic plasticity of rIBV and determine the pattern and frequency of viral polymorphisms during egg passage under immune pressure with polyclonal chicken sera and monoclonal antibodies specific for defined epitopes on S1. Newly emerged viruses will be deep sequenced and we will determine if antigenic shifts result in a shift in serotype.

Viral diseases are a constant threat to the poultry industry through reduction in broiler production, decreases in egg production & quality, and effects on animal welfare. IBV causes an acute highly contagious and economically important respiratory disease causing economic losses to the global poultry industry. Beyond the academic scientific community, the proposed research may also realise tangible benefits of a social and economic nature. These will be of benefit to The RI and TPI, the BBSRC and its stakeholders. The outcomes of the research will be of interest to other groups such as the Poultry industry, vaccine producers, DEFRA, veterinarians, students and the general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored webpages and press releases to the media. Overall the proposed research will have the following impacts.

UK economy: A 2005 DEFRA-funded report estimated that IBV affects 22 million birds in the UK, incurring and overall cost of £23 million per annum. 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. It has been estimated that every 10% reduction in IBV would be worth around £2.4 million to the UK Industry and £654 million globally. Infectious bronchitis was ranked by the commercial sector as the second most important disease of poultry in terms of the number of affected poultry between 2006 and 2009 and accounted for the largest segment (24.3%) of the poultry diseases market in 2012; this is expected to increase by 7.8% from 2013-2018.

BBSRC: Food security is a key research priority in the BBSRC Strategic Plan. Results from this project will provide crucial information as to why vaccines used to control an important avian endemic pathogen fail to induce cross-protection, on the use of existing vaccines and on the development of 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 to the global poultry industry. In 2012 the UK poultry meat industry sales were £6.1 billion; £3.3 billion contributed to UK GDP, with every £1 billion generating another £1.3 billion in the rest of the UK economy and supporting 73,200 jobs. Improved efficiency of the industry, through improved protection against endemic diseases such as IBV and the development of more efficient vaccines, particularly against continually emerging variants, will have positive benefits both socially and for the UK economy. Unravelling the mechanisms of cross protection will allow us to predict the combinations of vaccines needed to protect against multiple strains in the field, improving animal welfare, reducing losses to the poultry industry and risks to food security.

Pharmaceutical companies: Both groups (RI and TPI) have established collaborations, including direct support, with several vaccine companies that have resulted in ongoing assessment of potential vaccine candidates and immunomodulatory products. The data generated during this project will allow us to predict which combinations of the currently available vaccines are needed in the field to protect against current and newly emerging IBV strains.

Academia and Training: Results with respect to mechanisms of cross-protection, epitope prediction and the effect of immune pressure on virus mutation will be of interest to a wide scientific community and will be published in peer-reviewed journals and presented at national and international scientific meetings. The project will provide training in immunology, epitope mapping, molecular virology and bioinformatics.