Mapping fish CD4 T cell subsets for vaccine improvement

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This project involves collaboration between two teams of experts at the University of Aberdeen and University of Santiago of Chile, conducting state-of-the-art research in complementary areas of fish immunology/fish vaccination. Aquaculture is one of the fastest growing sectors that provide food to the expanding world population. It is estimated that ~50% of fish consumed worldwide are farmed, and this figure is projected to rise. Sustainability of fish farming relies on good management of fish health and control of diseases. Vaccination is an effective strategy to control many common diseases and many highly efficacious fish vaccines exist. However, the development of fish vaccines has been largely empirical, based on whether a formulation is effective at increasing survival post-disease challenge. This is unsatisfactory from both ethical and scientific perspectives. There is a clear need to establish methods to improve fish vaccine development.This project will undertake studies to characterise an important immune pathway that may be of vital importance for future fish vaccine development. Vaccination relies on the stimulation of adaptive immunity in vertebrates, with long-term memory responses giving protection when encounter with a pathogen occurs. In mammals a key effector population driving such responses are T helper (Th) cells, that release intercellular mediators (cytokines), that initiate antimicrobial responses, including antibody production. These responses have to be tailored to the pathogen type, with viruses, parasites and extracellular bacteria requiring different immune mechanisms to give protection. Different Th subpopulations differentiate in response to these different pathogen types and host factors, and release different repertoires of cytokines to produce the most appropriate response. We know virtually nothing about these responses in fish, although many of the genes involved in mammals are now characterised or have putative homologues likely to have equivalent function. For example Th cells express CD4 on their surface, and two types of this molecule exist in teleost fish, that will likely define these cells. Here we propose to develop antibody reagents to the two CD4 molecules to detect, isolate and characterise the CD4 subsets (CD4-1+, CD4-2+, CD4-1+/CD4-2+). In immunised fish, we will study their ability to express different cytokine repertoires upon restimulation in vitro with specific antigen. Sorted CD4 cell populations will be analysed. As antigen, both a bacterial and viral protein will be used in rainbow trout, an important farmed fish in both Chile and the UK. We have developed many reagents (eg primers for immune gene expression analysis) and immune proteins (eg recombinant cytokines) for use in trout, and expect the responses to be representative of those in salmonids more generally. Following the initial experiments, we will study the effect of adding different cytokines together with the specific antigen on the ensuing responses, by analysing cytokine gene expression and CD4 subset variations. We will next select the cytokines showing the most marked effects on directing these responses to link back to confirming the involvement of CD4 (putative Th) cells. This will be done by analysing cytokine gene expression in the sorted (CD4) cell subsets following antigen restimulation in vitro in the presence of the recombinant cytokines. These results will go a long way towards confirming the function of Th cells in fish, and will establish if they can express different cytokine repertoires in response to specific antigen and cytokines. We anticipate these responses will be of value as markers of protection in future vaccine development programmes, helping to improve the efficacy of poorly performing vaccines, and to generate vaccines to emerging diseases. They may also provide an alternative means to evaluate vaccine performance, reducing the numbers of fish undergoing pathogen challenge.