Development of novel oral vaccination s;trategies for Atlantic salmon

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci


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.

Technical Summary

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.

Planned Impact

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.
Description The research was undertaken in 3 work packages (WP).
In WP1 (Advancing knowledge of gut immunobiology in Atlantic salmon) the distribution of cells expressing markers for antigen presentation, and adaptive immune responses, in different gut regions, was determined using transcript analysis to key marker genes, and a range of available antibodies to key immune proteins. Next, gut associated lymphoid tissue (GALT) cells were isolated from pyloric caeca, mid gut and hind gut of trout and salmon, and were characterised in terms of their immune gene transcript profile and response to PAMPs and their ability to produce cytokines associated with innate immune responses and adaptive immunity. Cytokines related with pro-inflammatory responses were upregulated post LPS stimulation, interferons by poly I:C, and IFN-? and IL-4/13 were induced post PHA (a T cell stimulant) stimulation. Mucosal B cell (IgT) and T cell (CD3) responses were characterized in situ by IHC revealing differential distribution throughout the gut.
In WP2 (Identification of markers of protective immune response associated with oral vaccines) commercially available vaccines used for oral boosting in fish (Vibrio, ERM) were evaluated for their effectiveness, and in parallel the genes modulated in different gut regions following oral delivery were studied. In this WP a panel of potential markers associated with protective immunity were identified. Mucosal antibody, IgT, expression for example was consistently upregulated in oral boosted rainbow trout, whereas upregulated TCR expression appeared to be significantly greater in immersion boosted fish. Proteomic analysis by quantitative DIGE and MALDI PMT analysis of hind-gut from the ERM vaccination trial revealed alterations to relative protein tissue profiles at days 12 and 29 post-boost vaccination and at 1 day post-challenge. Analysis of identified differentially expressed proteins in fish under different regimes (immersion prime, no prime/oral boost, or immersion prime/oral boost) associated key functional activities primarily to cellular metabolism and immune system processes. Proteomic analysis was similarly performed on pyloric caeca samples from Vibrio vaccination trial groups (non-vaccinate, immersion prime/oral boost, immersion prime/immersion boost) immediately pre-challenge. High variability in protein expression within individual pyloric caeca tissue was observed with minimal variation between groups. N-lysine methyltransferase was found to be significantly elevated in both immersion prime/oral boost and immersion prime/immersion groups, and pyridoxal kinase-like down-regulated in the immersion prime/oral boost group.
In WP3 (Vaccine delivery using nanoparticles- Novel salmon oral vaccination using silicon-based nanoparticle technology) two vaccines were studied, for furunculosis and SAV. Initially formalin-inactivated Aeromonas salmonicida was incorporated into different silicon nanoparticle (NP) formulations by SiSaf (our industrial partner). In vitro assessments of these formulations were undertaken by testing antigen stability in acidic environments (mimicking the fish gut environment), and by stimulating RTS-11 (monocyte/macrophage) cells, head kidney and gut cells. It was found that pro-inflammatory and adaptive cytokine gene expression increased following stimulation of these cells, and two formulations were selected to be tested in vivo: FD3 (which gave the best results in terms of gene expression) and FD1 (which gave good results in terms of gene expression and was the formulation with the greatest antigen stability). In vivo trials using oral delivery of these two formulations were undertaken, examining different antigen doses, administration schedules and tissues. Results from both trials revealed a wide range of immune gene (transcript) modulation in the gut. In addition, the studies showed for the first time that oral (silicon) NP delivery was safe for fish (with pilot studies performed following discussion with the HOI). Oral vaccination against furunculosis was subsequently undertaken with two formulations, with intraperitoneal (IP) injection used in parallel, to assess protection. Whilst no clear beneficial effects were seen with oral administration with the A. salmonicida antigen preparation, the injected A. salmonicida NP gave a degree of protection and no side effects were seen. Oral gavage experiments of Atlantic salmon using GFP-labelled NP antigens revealed early induction (24-48 h) of cytokine markers identified from earlier studies in trout. This study also highlighted the stability of NP formulations after >10 month storage at 4°C. For the SAV studies two trials were performed. In the first, fish were vaccinated with different combinations of commercial pancreatic disease (PD) vaccine and oral NP formulated vaccine followed by oral NP boost. The results of trial 1 were promising and demonstrated enhanced protective efficacy of vaccination when a NP oral boost was administered to either a NP oral challenge or a commercial IP challenge. In this trial, significantly higher average weight was observed in NP boosted fish compared to other groups including commercial IP vaccine alone. Viral loads and numbers of virus positive fish, as determined by qPCR, were significantly lower in these groups compared to other treatment groups. Cytokine profiling of a range of tissues suggested potential immune modulation through NP boosting. Significant in vitro and in vivo changes in cytokine transcription were observed with NP boosting (4 days post-boost), including upregulation of IFN-? and IL-6 and significant upregulation of TGF-B compared to commercial IP control. In addition, fish in these two groups exhibited no behaviour related-effects associated with SAV-disease and remained normal and active throughout the study. A number of potential correlates of immunity were revealed through proteomic analysis of both head kidney tissue and serum. In trial 2 fish were primed with conventional SPDV IP vaccine or NP formulated vaccine administered IP (time point 1). Fish were boosted 4 weeks later (time point 2). Boosts were either in-feed oral vaccine antigen formulated with NP or non-formulated antigen delivered orally. In this trial, the exposure to SPDV did result in positive heart tissue but samples were weak as tested by qPCR. However, all vaccine groups showed lower numbers of qPCR positive fish after challenge indicating that there was some protective effect. In addition, all vaccine groups showed lower virus neutralisation scores compared to the negative control (unvaccinated) group. Contrary to the first study there were no significant differences between groups in terms of body weight at any time point.
In summary, NP delivery to fish has been shown to be safe, and the NP were effectively loaded with bacterial and viral antigens for oral or IP administration, and these preparations showed long term stability. Oral priming for furunculosis was not effective but IP priming looked promising. With SAV oral boosting with NP gave enhanced protection, following oral or IP priming, associated with modulation of a range of cytokines. A second trial also showed benefit from boosting with NP in-feed although this challenge was not as discriminatory between groups.
Exploitation Route We will use these findings to aid future oral vaccine development for fish. This programme (ARC) has links to appropriate industry connections to allow rapid translation of any interesting results.
Sectors Agriculture, Food and Drink

Title Use of silicon nanoparticles for antigen delivery in fish 
Description We have verified that it is safe to use silicon nanoparticles as a delivery method in fish, specifically trout and salmon. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact A paper will be published on this approach for vaccination, that will allow other researchers to assess this method for a variety of applications. 
Description Fish Immunology conference (Portland, Maine) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A poster has been accepted for the International Society of Fish & Shellfish Immunology conference in Maine, for June 2016, entitled "Immune gene profiling of different gut regions and gut associated lymphoid cells from rainbow trout (Oncorhynchus mykiss). Presented by Dr Sohye Yoon.
Year(s) Of Engagement Activity 2016
Description Mucosal nanoparticle immunisation against salmon alphavirus (SAV) in Atlantic salmon (Salmo salar L.). 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact A poster was presented at the European Association of Fish Pathologists Conf., Porto, September 2019.
Year(s) Of Engagement Activity 2019
Description Poster(2) at ISFSI mtg in 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A poster entitled "Immune gene profiling of different gut regions and gut associated lymphoid cells from rainbow trout (Oncorhynchus mykiss)" was presented at the ISFSI conf. in Portland, Maine, in June 2016.
Year(s) Of Engagement Activity 2016
Description Talk at EAS in 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk entitled "Immune gene profiling of different gut regions and gut associated lymphoid cells from rainbow trout (Oncorhynchus mykiss) and immune responses following oral vaccination against Yersinia ruckeri." at the European Aquaculture Society meeting in Edinburgh in September 2016.
Year(s) Of Engagement Activity 2016
Description Talk at the EAFP meeting in Belfast, September 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk entitled "Immune reactivity of isolated Atlantic salmon (Salmo salar) gut leucocytes to PAMPs and recombinant cytokines."
Year(s) Of Engagement Activity 2017
Description The development of novel nanoparticle based oral vaccines for use in salmonids. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk at the Int. Soc. Developmental and Comparative Immunology Conference, in Santa Fe, USA.
Year(s) Of Engagement Activity 2018
Description Vaccinology workshop (Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A talk was given on the on-going work to the ARC workshop in Edinburgh in February 2016, in a talk entitled "Development of novel oral vaccination strategies for Atlantic salmon". Presented by Dr Sohye Yoon.
Year(s) Of Engagement Activity 2016