Development of novel oral vaccination s;trategies for Atlantic salmon

Farmed salmon is Scotland's number one food export with a retail value >£1billion worldwide, and Scottish salmon is
exported to over 60 countries. The Salmon aquaculture industry employs over 2,200 people and has invested over
£205million between 2006 and 2011, making this industry a major player in Scotland's economy. The world's increasing
demand for animal protein has pushed the consumption of farmed fish from 9% of total fish consumed in the early 80's to
nearly 50% at present, and this is likely to increase further in the coming decades. Producing enough farmed fish to supply
this demand will only be possible if the major bottlenecks to increased production are reduced or removed, and this includes
the control of infectious diseases. Salmon are fish that require a high quality environment for optimal growth but even when
this is provided, occasionally diseases will arise and thousands of pounds can be lost due to fish mortality and quality
depreciation. Fortunately in the late 1980's a strategy for disease prevention was established: fish vaccination. This
strategy has been so successful that the use of antibiotics in aquaculture has almost disappeared and all the salmon that
are farmed in Scotland will have been vaccinated at least once in their life. Although very successful this strategy has two
main drawbacks: the need for individual fish vaccination and the side-effects of adjuvants included in the vaccine
formulations. In 2011 nearly 50 million fish were vaccinated in Scotland alone. This is a very costly process and in addition
causes a significant amount of stress that makes fish susceptible to other diseases and can only be employed before they
are moved to sea. The side-effects from oil-based adjuvants usually include localized inflammation within the peritoneal
cavity, which can compromise growth and depreciate the fillet value. To overcome this problem we propose to undertake
research to allow development of oral vaccination for salmon. This will include testing of a novel oral vaccine delivery
technology based on nanoparticles. Oral vaccination has several advantages in comparison to injection: 1) the vaccine
would be formulated into the feed, making it easy to administer, 2) the stress of handling the fish is thus avoided and the
need for chemical treatments post-vaccination, to prevent opportunistic pathogens, is avoided, 3) extra doses can be given
after fish have been moved to sea. Therefore this technology will help make salmon farming more efficient,
sustainable, and reduce the cost of disease prevention. To achieve this goal we will start the project with basic research
on how foreign molecules are recognized and presented to immune cells in the gut of salmon, key knowledge required to
understand oral vaccine efficacy. We will then elucidate some gene markers of vaccine effectiveness using existing
commercial vaccines that use mucosal delivery, either by immersion of fish in the vaccine solution as a primary
vaccination, or given as oral boosters. Lastly we will evaluate the use of a novel technology, silicon based nanoparticles,
to deliver vaccines against two commercially relevant diseases for Atlantic salmon: Furunculosis and Pancreas Disease.
With the completion of the salmon genome we know more about fish immunity than ever before. This project will use this
knowledge to undertake ground breaking research on several aspects of gut immunity and oral vaccination, helping the
UK's aquaculture industry to remain sustainable and to continue to grow over the coming years.

Oral delivery of vaccines to fish has been problematic, mainly due to gut processing of antigens. However, commercial oral booster vaccines exist for trout for ERM and vibriosis, and extend immunity from priming by other routes. In this project we aim to extend the knowledge base of gut immunology, and characterise markers of effective vaccination to aid future oral vaccine development. In addition we will use a new technology for oral delivery, orthosilicic acid nanoparticles (OAN), and establish its efficacy in fish, aided by the advances in gut immunology from this project.

First we will define the gut regions in salmon responsible for recognising orally-delivered antigens and create a detailed immune profile. We will use qPCR and in situ hybridisation (ISH) to study immune transcripts, and immunohistochemistry (IHC) with pre-existing antibodies to study immune proteins. We will also isolate intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) and stimulate with PAMPS and cytokines to study their responses and survival, and assess the impact of these molecules in the hind gut after anal intubation.

Next we will use commercial oral vaccines to study protective immune responses in the gut, initially in trout (as they are trout vaccines) but then in salmon, to allow a comparative analysis. We will use transcriptomic profiling (qPCR and microarrays), ISH and IHC, analysis of induced specific antibody and proteomic/metabolomic analysis, the latter to detect molecules linked to gut protective immunity in an untargeted way.

Lastly, we will evaluate the OAN for delivering a bacterial vaccine to Aeromonas salmonicida, since the failure of immersion and oral vaccines for this disease has driven the need for injection vaccines containing oil adjuvants, and to Pancreas Disease, an alphavirus that is a major disease of salmon. This will also give an initial assessment of the markers identified, in the context of a new oral vaccine formulation.

The development of novel oral vaccination strategies for Atlantic salmon will have long lasting economic and societal benefits by increasing the sustainability and growth of the UK fish farming industry. Beneficiaries will include aquaculture, government, private enterprise and, ultimately, the wider public as consumers. Aquaculture currently provides nearly half of all fish consumed globally and is one of the fastest growing animal-food producing industries. Scotland is the third largest producer of Atlantic salmon and in 2012 production reached 162.2 tons, making it the largest food export with an annual retail value of > £1 billion. With the human population expected to rise 61% by 2100, and as over-fishing continues to reduce wild fish stocks, aquaculture will be placed under considerable pressure to meet consumer demands. The production and promotion of fish products rich in EFAs (e.g. omega-3) and high quality protein is also required to tackle public health issues and improve consumer awareness. As fish farming continues to expand and intensify, however, the frequency and severity of disease outbreaks will increase if effective control measures such as vaccination are not in place. Disease can be catastrophic for the industries viability as high mortality rates ultimately lead to severe financial losses, site closures and redundancies. There are also environmental, ecological, animal welfare and human-health concerns following disease outbreaks.
Treating disease in aquaculture has proven difficult. Excessive use of antibiotics as the industry emerged led to antimicrobial resistance genes within bacterial populations. Currently the use of antibiotics in UK aquaculture is low compared to other farmed animals due to the success of existing bacterial vaccines and it is important that this is sustained. Although emphasis has been placed on prophylaxis, primarily vaccines, to successfully prevent major epizootics the current vaccines for salmon are not ideal, as most are administered via intraperitoneal (IP) injection. This is costly and labour intensive and can cause collateral losses either by favouring infection with opportunistic pathogens (e.g. Saprolegnia) or from side effects of the vaccine itself (e.g. adhesions). IP injections are known anthropogenic stressors which can lead to an immunocompromised state, whereas improper application can result in fish death. Thus IP injections of vaccines are a major concern for animal welfare, and as many vaccines are adjuvanted, pose as a major occupational health risk to fish farmers. IP vaccines are only viable during the freshwater phase of salmon farming and thereby limit the vaccination timeframe. Developing an oral antigen delivery technology would address many of the problems associated with IP injection with the potential to 1) decrease the costs of fish vaccination, 2) expand the window for vaccination, 3) reduce losses associated with side effects and/or opportunistic infections, and as a result 4) improve animal welfare. Given the importance of the Atlantic salmon industry to the UK economy, both the UK and devolved Scottish governments will have a shared interest in developing new oral antigen delivery technologies. The Scottish governments National Marine Plan, which aims to achieve a 50% increase in aquaculture production by 2020, will require a significant technological innovation of this kind if it is to ensure sustainability and prevent disease. Initiatives supported by the UK government to discover and apply new technologies to improve animal welfare (i.e. NC3Rs) will profit from this research. Through close collaboration with the global animal health company MSD, one of the main suppliers of fish vaccines, findings will have a direct route to commercialisation. Given that the technology will be transferable to other fish species, it has broad long-term commercial prospects. Overall, this project will help maintain the UK as a world leader in salmon farming.