Vaccines for chronic viral pathogens in salmon- generation of interferon attenuated cell lines

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

Abstract

Vaccines for chronic viral pathogens in salmon- generation of interferon attenuated cell lines.
Acroym: SalVacCell

Project goal / key aims.
There is a major requirement to generate new vaccines for viral pathogens in the salmon aquaculture industry. Viruses represent one of the major economic losses to the salmon industry, which is a direct reflection of the lack of highly protective vaccines. In order to improve vaccine design and testing high quantities of viruses are required, but at present this is not possible. This project will use cell lines that have been edited by CRISPR/cas9 to be highly permissive for growth of difficult to grow viruses. We will knockout the type I interferon pathway which is the major antiviral mechanism in animals. Specifically we will target key genes that regulate interferon induced cellular responses to viral infection. These cell lines will additionally be used to explain the underlying function of genes associated with natural viral resistance / susceptibility in salmon that is of direct importance to breeding programs.
We have secured Industrial partners Benchmark (vaccine company) and Landcatch (fish breeding). Consortium agreements will clearly define roles and confidentiality of IP.

OBJECTIVES
1. Development of IFN-deficient fish cell lines by knock-out of IFN function
2. Evaluation KO cell lines to mount an antiviral response to confirm phenotype
3. Use the cell lines to explain naturally occurring resistance / susceptibility to fish viruses
4. Comparison of yield for viral particle production and viral diagnostic turn-over time between traditional cell lines and newly developed cell lines

KEY CHALLENGES

The overarching challenge is to be able to produce high titres of virus for vaccine companies to be able to improve design and protection to economically important chronic viral pathogens. Many labs have attempted to knock down genes in salmonid cells, but to date this has not been achieved. To our knowledge we are the first to create such cell lines and as such our unfunded preliminary work has already overcome a major hurdle. The challenge of this project is to fully exploit our cell line technology for both disease management and also to greatly improve the basis of genetic selection.

Deliverables- to be used by partners

a. Engineered cell lines that are deficient in antiviral responses.
b. Cells lines that can be used to produce high titre of viruses for vaccines
c. Capacity to upscale viral production in industrial environment.
d. Improved precision of selection for disease resistance in salmon breeding

Project duration: 24 months

Total project cost: £354622

Contribution requested from (100%) BBSRC/NERC £249,622 (£199697.60 80%)

Contribution from MSS: secured £65K
Contribution from Benchmark secured £20K
Contribution from Landcatch secured £20K

Planned Impact

Our main goal is to use recently developed salmonid cell lines using the CRISPR/cas9 methodology to edit genes and alter phenotype of cells, specifically to provide enhanced methods for control of viral infections in farmed salmon. Two approaches will be taken 1. To enhance vaccine production and 2. Improve natural resistance to virus infection. The cells will be further modified with our industrial partners to improve vaccine production for viral pathogens that are currently unavailable or have limited effect. Secondly the cell lines will be used to improve selective breeding approaches by targeting candidate viral resistance loci already identified by classic breeding programs and genome wide association studies.
Our key aspiration is to reduce the economic losses that results from viral infections in aquaculture, vaccines are the best choice for control, but vaccine design and the ability to grow certain viruses in cells has retarded production. Some economically important viral pathogens result in major economic losses to the UK aquaculture industry, some of these viruses do not result in death of the fish but reduce performance and final product quality termed chronic viral infections. In parallel it is highly likely emerging viral pathogens or more virulent strains may appear at any time and our approaches will ensure vaccine trails and challenges can be performed much faster.
Using the CRISPR-CAS9 gene editing technology, genes involved in natural resistance to viruses can be studied in vitro. Currently, marker-assisted selection is used in breeding programs to improve disease resistance. Research will lead to the discovery of more accurate tools for selective breeding of fish with better disease resistance. This will be possible due to selection based on causal genes directly, rather than linked markers. The output of the project will benefit the aquaculture industry as the two industrial collaborators, BenchMark and Landcatch, are key players in vaccine development and fish breeding. In the long run, the project will help maintain the UK as a world leader in aquaculture.
The project has two main areas for commercial and academic impact, firstly cell lines for vaccine production and secondly for underpinning selective breeding. The project partners will meet twice per year with at least one industrial partner at the meeting. A regular part of the meeting agenda will be to review knowledge and expertise exchange. We do not anticipate both industrial partners being present together at all meetings and a consortium agreement will ensure any possible areas that there is commercial competition will be clearly defined.
To reach additional audiences, our industrial partner Benchmark is a gold sponsor of the biannual "International conference on fish and shellfish immunology", while Landcatch sponsor sessions at major international breeding conferences, such as the World Congress of Genetics Applied to Livestock Production. Prof. Martin is a forum member for the aquaculture theme within The Marine Alliance for Science and Technology for Scotland (MASTS) and was also a member of the Scottish Government Ministerial Group on Sustainable Aquaculture. In addition to dissemination of results in scientific journals and meeting, we will target trade press releases and presentation at conferences attended by the UK aquaculture industry (e.g. Aquaculture UK). These are both avenues through which the impact of our project targets key governmental, industrial and strategic plans for aquaculture research.
The innovative aspects of the project will lead to additional uses for the cell lines beyond the project and publications in high ranking journals to ensure academic impact of the project when the outs are not commercially sensitive. The outputs of this project will also help to meet the 3Rs by improving both vaccine production, disease trials and for improved knowledge related to selective breeding.

Publications

10 25 50
 
Description There are many viruses that infect fish in the aquaculture industry that cannot be grown in cell culture, this means that high titres of viruses cannot be obtained for use in vaccine production (main theme of cells). In addition pure virus for fish challenge experiments is not available, which these cells would allow, thirdly the cells could be used for diagnostic purposes where confirmed diagnosis is required. We have engineered a cell in which the interferon pathway is Gene editing has destroyed. Interferons drive the antiviral activity in cells, as such these cells may have the potential to be permissive to growth of viruses that cannot be done in other fish cell lines.
Exploitation Route The cell lines colud be used comercially for vaccine production, but would need to have some further refinements. they could also be used for viral diagnostics in aquaculture
Sectors Agriculture, Food and Drink

 
Description Interferons (IFN) belong to a group of cytokines specialised in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of Interferon Stimulated Genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFNs and type II IFN (IFN?), but no type III (?) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, since alternative chains are used in type I IFN receptors. The mechanisms of the downstream signalling remain partly undefined. In mammals, members of the Signal Transducer and Activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signalling. In fish, its role in IFN signalling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defences in fish.
First Year Of Impact 2017
Sector Agriculture, Food and Drink
Impact Types Societal

 
Description Improving resistance to infectious salmon anaemia using genome editing: Novel approaches to tackling viral disease in aquaculture
Amount £205,540 (GBP)
Funding ID BB/R008973/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Title Gene editing 
Description Gene editing in cell lines: a cell ine has been developed that can be used to generate clonal gene edited cells. This is based on editing in tandem both GFP and target genes. This approach is being used in the project to interfer with viral uptake and replication. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact A paper was published denomstrating the gene editing technology in salmonid cell lines. These cel lines based on Chinook salmon embyro cells have been stabily transfected with both green fluresence protein and cas9 plasmid. 
 
Description INRA, Fish Virology and Immunology. Dr Collet and Dr Boudinot 
Organisation French National Institute of Agricultural Research
Department INRA Versailles
Country France 
Sector Public 
PI Contribution The Lab in INRA is also working on Gene editing in fish cell lines.
Collaborator Contribution Exchange of knowledge and approaches for gene editing
Impact None as yet
Start Year 2017
 
Description Collet B, Lester K, Zou J, Dehler C, Boudinot P, Martin S (2017). Type I interferon signaling in a salmon cell line with a CRISPR/Cas9-mediated disrupted signal transducer and activator of transcription (stat)-1. 18th European Association of Fish Pathologist Conference (EAFP), Belfast, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International Conference presentation
Year(s) Of Engagement Activity 2017
 
Description Collet B. (2017). Engineering cell lines for fish health research. 18th Fish Immunology Workshop, Wageningen, the Netherlands. 30th April - 4th May. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Postgraduate workshop on Fish immunology, Wageningen, The Netherlands
Year(s) Of Engagement Activity 2017
 
Description Sam Martin Interferons and cell lines for vaccine development. CIFRI Platinum Jubilee Lecture, Central Inland Fisheries Research Institute, Kolkatta, India. 20th June 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited international seminar in India
Year(s) Of Engagement Activity 2017
 
Description Sam Martin, Carola Dehler & Bertrand Collet. (2017). Gene editing by CRISPR/cas9 for generating interferon attenuated fish cell lines. Marine Alliance Science and Technology Scotland. Annual Science Meeting, Glasgow, September 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Conference presenation
Year(s) Of Engagement Activity 2017