Immune cell dynamics predictive of vaccine protection in Atlantic salmon

Salmon farming is a key UK food sector, producing more than 200,000 tonnes of fish in 2019, representing the top national food export. This sector provides an economic contribution of £1.8-billion per year and is responsible for around 8,000 jobs, concentrated in rural regions of Scotland. The salmon industry has ambitious growth targets, aiming to double its contribution to the economy and job market by 2030. This ambition is hindered by negative impacts caused by infectious disease to fish welfare and the environment, with a range of viral diseases being a major current problem. To help the sector grow sustainably, such challenges need to be overcome with the support of scientific innovation.

Viral disease outbreaks on salmon farms cause major financial losses and welfare problems. There further exists a continuous threat of new viral diseases entering aquaculture systems due to climate change. There are no treatment options to control salmon viral diseases, and vaccination remains critical, with several viral vaccines on the market for commercial use. Over 50 million fish are vaccinated in the UK each year. While this effectively controls bacterial diseases, viral disease remains an unsolved problem. Consequently, the development of new and improved vaccines to control viral diseases in farmed salmon remains critical.

Developing effective vaccines requires extensive fish use, with hundreds of animals killed per trial to gain data on efficacy after disease challenge. The whole process is expensive and time consuming. Methods that accelerate vaccine development, while substantially reducing the number of animals killed, will represent an important step forward in terms of animal welfare and cost savings.

Fish share many components of the immune system with mammals leading to protective responses following vaccination. However, we have a poor understanding of the role played by the large diversity of different immune cell types in protective immune responses and immune memory in fishes. New technologies allow us to quantify global gene expression in individual cells (so-called 'single cell transcriptomics') and hold exceptional promise to transform our understanding of cell diversity responsible for immune protection following vaccination in fish.

Our aim is to create a large up-step in understanding of how vaccination against a major problem viral disease (called pancreas disease) leads to immune protection through dynamic changes in immune cells. We will apply single cell transcriptomics to samples taken from a carefully designed vaccination experiment, designed to link early changes in immune cell expression to immune protection generated over months. The first major aim is to describe the full diversity of different cell types in the major tissues of the salmon immune system, including how these cells respond to viral vaccination. We will compare two registered pancreas disease vaccines with very different formulations to understand cellular mechanisms leading to differences in disease protection. The second major aim is to identify immune cell types, and specific marker genes for these cells, correlated with vaccine protection. A final objective is to use the results to develop a cost-effective platform that can be used to accurately predict disease protection early post-vaccination.

In addition to creating major knowledge advancement on the cellular basis of protective immune responses in fish, the results will have applications in vaccine research and development, opening up strategies to assess vaccine efficacy using faster more cost-effective strategies and fewer animals. This may lead to improvements in vaccine design and testing that can positively impact the sustainability of salmon aquaculture while promoting fish welfare.

This project addresses the need for new and improved vaccines in Atlantic salmon, with emphasis on early determination of efficacy. We will reconstruct immune cell lineage temporal responses to vaccination at unprecedented resolution using single cell transcriptomics and apply the knowledge to help predict disease protection outcomes early post-vaccination. As a model, we will compare two distinct vaccine formulations to pancreas disease (PD) caused by salmonid alphavirus (SAV), which remains a major problem in aquaculture. A naïve cohort of fish will be split into three treatments: i) monovalent PD vaccine, ii) multivalent PD vaccine, and iii) PBS control (intraperitoneal injections). Fish will be split into non-lethal repeat sampling and lethal sampling groups, and sampled at common timepoints post-vaccination, before PD challenge and a final sampling for PD pathology. Head kidney, spleen, blood and peritoneal cavity leukocytes will be sampled to capture temporal development of the cellular immune response by single-nuclei RNA-Seq (snRNA-Seq). We will assess humoral responses by measuring development of antibodies to SAV. Our study design allows for assessments of PD pathology (heart, muscle and pancreas lesions; viraemia, tissue viral load) and antibody data to be correlated with snRNA-Seq data on an individual level. We will generate >1-million single-nuclei transcriptomes spanning 168 samples (50,000 reads per nucleus). snRNA-Seq data will be analysed to describe immune cell lineages/subsets and their responses to the PD vaccines, with trajectory inference used to describe B and T cell differentiation pathways. We aim to identify immune cell lineages and cell-specific marker genes correlated with PD protection across the post-vaccination timecourse. Finally, we will develop, validate and benchmark a unique Fluidigm BioMark qPCR array for high-throughput, cost-effective measurements of cell-specific markers that correlate strongly with PD vaccine protection.