Development of a proteomic platform to facilitate the generation of new and improved vaccines for use in aquaculture.

With concerns over dwindling wild fish stocks and the UK government recommending that we all eat two portions of fish a week as part of a healthy diet, we are increasingly turning to aquaculture, the farming of fish and shellfish, as a sustainable way of filling our needs. Over the last 20 years, aquaculture in the UK has developed into an industry worth well over £1 billion per year, dominated by Scottish salmon farming. To ensure that the industry can meet increasing demands for sustainable fish, the government is aiming to grow UK aquaculture production 25% by 2020.

Infectious diseases are the most significant threat to the stability and future expansion of the aquaculture industry; much like in human cities, occasionally previous bacterial and viral infections can re-emerge in a fish farm or new infections can spread from other places. An infection outbreak can cause massive financial losses due to fish death, costs of expensive interventions, or the poor quality of the resulting fish flesh. Also, as for us humans, the best way to prevent disease outbreaks on fish farms is by vaccination; this strategy is so effective for bacterial diseases that the use of antibiotics in aquaculture has almost stopped completely. In fact, every salmon farmed in Scotland will have been vaccinated at least once in its life.

While very successful for some diseases, fish vaccination in its current form also has a number of drawbacks; primary among these is that each fish has to be individually injected with a vaccine, which is quite a challenge considering more than 150,000 tonnes of salmon are produced each year! This is not only costly and time-consuming but can cause the fish to become stressed making them susceptible to other opportunistic infections. Second are the potential side-effects of the immune system stimulants (or 'adjuvants') present in the vaccination; the optimal formulation will have adjuvants strong enough to induce a robust immune response but not so strong that they cause side-effects impacting the quality or welfare of the fish. Finally, some diseases have proven more challenging than others in terms of developing effective vaccines.

For these reasons, many scientists are trying to find better ways to administer fish vaccines and adjuvants, while looking for new ways to vaccinate against fish diseases where no vaccine yet exists. However, vaccine development and validation is a slow process that requires extensive scientific testing with living fish. Therefore, there is great interest in the development of approaches that will reduce the number of fish required for vaccine testing, while making the testing process more robust at the same time. With this in mind, our project aims to adapt a new 'proteomic' technology currently used in the study of human disease - to quickly and accurately monitor fish immune responses. The method allows extremely precise measurements of protein levels and will allow us to accurately monitor key factors involved in an effective immune response such as antibodies. Our approach will allow miniscule blood samples to be taken from the same fish many times during an immune response, which is an improvement on comparable existing methods that require much more blood and hence a lot more fish to be sacrificed during an experiment.

Overall, our approach will enable scientists in the aquaculture sector to accurately monitor changes in fish immune protein levels in response to new and existing vaccines - allowing them to gauge the strength of immune responses and to predict the level of immunity conferred, whilst using fewer fish than current testing protocols. This in turn should help new vaccines and novel methods of administration to come online much more quickly, which will feedback to have positive effects on the sustainability and growth of aquaculture in the UK and worldwide.

Aquaculture is the fastest expanding sector of food and animal production in the UK and this growth is threatened by emergent or existing diseases. As the industry is worth >£1 billion per annum, considerable effort is being directed into development of new and/or more efficacious fish vaccines with easier, less labour-intensive methods of administration. To evaluate immune protection, most research groups currently use a vaccination-challenge strategy with terminally acquired samples, combined with quantitative PCR measurements of immune gene mRNA levels for validation. Such approaches require large numbers of animals to obtain sufficient statistical power and provide only limited information on the nature and kinetics of the protective response. Therefore, the objective of this project is to develop a proteomics platform that will allow rapid, repeatable and accurate quantification of multiple proteins in minimally-manipulated fish plasma samples. To achieve our objective, we will develop targeted mass-spectrometry (TMS) methods exploiting a state-of-the-art Hybrid Quadrupole-Orbitrap system. TMS proteomics is currently used mostly in clinical research and drug development and offers a number of routes to routinely quantify targeted proteins in complex biological samples down to ng/ml levels with high consistency and accuracy. Following optimisation, our final protocol will be combined with a non-lethal sampling technique allowing quantification of target immune proteins in plasma samples from individual rainbow trout over the course of an immune response. Our platform will enable scientists in the aquaculture sector to gain a more accurate and complete understanding of the immune response when testing new vaccines, adjuvants or modes of administration. In turn, this should shorten the time it takes for new and improved vaccines to come to market, while dramatically reducing the number of animals required relative to current testing protocols.

This study will bring together experts in the fields of immunology, bioinformatics, proteomics and monoclonal antibody production to address one of the main limitations to the expansion of the UK aquaculture industry- the need to rapidly develop new/improved fish vaccines that can be administered quickly to protect stock from infection. We anticipate our research will have a variety of academic, industrial and economic impacts, in the UK and beyond, culminating in benefits to the wider public:

Scientific community: the most immediate impact of this work will be within the international group of researchers working on fish immunology and vaccine development. The ability to routinely and accurately measure immune proteins in plasma samples by targeted mass spectrometry (TMS) will allow scientists in academic and commercial sectors to gain a more accurate and comprehensive understanding of fish immune responses when testing new vaccines, adjuvants or modes of administration. The method can be easily adapted to blood markers indicative of other traits of interest in aquaculture, for example growth or stress. We anticipate that many researchers working in others areas of biology and with other species will also benefit from the TMS protocols we develop; in fact, in the post-genomic era, where costs of obtaining sequence data are relatively low, there are almost no limits to the potential uses of TMS for molecular studies in both model and non-model organisms, including several research areas prioritised by NERC and BBSRC. Thus this project will play an important role in promoting TMS outside of its current clinical applications.

Aquaculture industry: disease outbreaks can be catastrophic to the aquaculture industry; high mortality rates lead to severe financial losses, site closures and redundancies. The TMS platform developed in this project and follow up work should enable new and improved vaccines to come to market more quickly, thus helping to remediate costs of disease to aquaculture in the long-term. Scientists in the aquaculture sector will also be interested in TMS as a novel tool for diagnostics (e.g. to gauge physiological status of the fish) and/or to rapidly screen fish for infection (e.g. by detecting viral or bacterial proteins in a sample).

Government: given the importance of aquaculture to the UK economy, the UK and devolved Scottish governments have a shared interest in the industry's expansion and stability. However, in order to achieve government objectives to increase production by 25% by 2020, new and improved fish vaccines and administration methods will be needed. The development of TMS will provide essential information to enable this process. Our use of non-lethal sampling for this study will also encourage other groups to use the same technique, supporting government initiatives (i.e. NC3Rs) to reduce the number of animals used for research.

Students: we have a large undergraduate, post-graduate and post-doctoral community within the College of Life Sciences and Medicine at UoA; this project offers the opportunity to introduce them to an emergent technology and discuss its adaptation to new areas of research.

Wider public: With concerns over dwindling wild fish stocks and increasing consumer demand for sustainably sourced fish, the aquaculture industry is under significant pressure to meet consumer demands. By facilitating the efficiency with which new vaccines can be tested and validated, our project will help the industry expand and meet these needs. Another area of public concern is the excessive use of antibiotics in the food production industry, a practice thought to have contributed to the emergence of antibiotic resistant bacterial strains. Due to the success of current fish vaccination programmes the use of antibiotics in UK aquaculture is low compared to other farmed animals, however the development of additional vaccines for fish bacterial diseases will ensure this continues.