Mapping fish CD4 T cell subsets for vaccine improvement

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


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 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

This project will undertake studies to characterise an important immune pathway that may be of 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 antibodies 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 recombinant cytokines. These results will go a long way towards confirming the function of Th
cells in fish, and will establish if they 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 nos of fish undergoing pathogen challenge.

Planned Impact

Eating fish has many health benefits but with the human population increasing and with wild fish stocks
harvested to capacity (or decreasing), aquaculture has to bridge the gap and meet consumer demands for fish. Indeed,
aquaculture currently provides nearly half of all fish consumed globally and is one of the fastest growing animal-food
producing industries. In the context of the present call, Chile and Scotland are the 2nd and 3rd largest producers of Atlantic
salmon globally. Chile is also Top 15 in global aquaculture. In 2013 production in Scotland alone reached 163.2 tons,
making it the largest food export for Scotland with an annual retail value of > £1 billion. Whilst in 2014 (after the 2007
collapse suffered by the ISAV outbreak) Chile was ranked again as the second largest producer of farmed salmon, with
revenues of US $ 5.63 million and representing about 30% of world production. The contribution to the economy and to
rural employment is recognised in both countries, as is its valuable contribution to nutrition and food security.
As fish farming continues to expand/ intensify, the frequency and severity of disease outbreaks have the potential to
increase if effective control measures such as vaccination are not in place. Although many effective bacterial vaccines
exist, vaccines for viral diseases are lacking or have lower efficacy, and vaccines still need to be developed for many
emerging diseases. Sustainability of the farming industry depends on a good health status.
This research programme will increase knowledge on the mechanisms underpinning establishment of protective immunity
by vaccination. If successful, and new ways to monitor vaccine effectiveness can be developed in the future, then many
benefits will likely materialise, with beneficiaries in aquaculture, government, private enterprise and, ultimately, the
consumer. Better ways to optimise vaccine formulations to elicit appropriate immune responses will aid vaccine companies,
and increase the speed at which new vaccines appear on the market, which will increase profits for both Pharma and fish
farmers via better health management. Fewer losses during farming will increase production, improve animal welfare and
potentially increase employment, especially in rural areas. The technological advances seen in salmonid farming will likely
spread to other species in other countries, with fish farming an important industry in many developing countries. In addition,
immune measures of protection have the potential to reduce the number of fish being exposed to pathogens as part of
vaccine development and vaccine potency batch testing. Such methods could be extrapolated from the data obtained in
this study should it become clear that Th cells exist in fish. In addition, as Chile has a duty to reduce the use of antibiotics in
the salmon industry, knowledge and methodologies leading to improved current vaccines will help to accomplish this
important task.
The Scottish governments National Marine Plan proposes a 50% increase in aquaculture production by 2020, and this will
require significant technological innovation 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 our close links to global (fish) vaccine companies (eg Zoetis, MSD, Elanco, Pharmaq), our findings will have a
direct route for translation to benefit vaccine development programmes. Given that the methodology could be transferable
to other fish species (i.e. for fish species within the aquaculture diversification program in Chile), there is the potential for
long-term commercial gain if successful. Thus, ultimately this project aims to help maintain the UK and Chile as a world
leader in aquaculture and aquaculture research, whilst developing methods that may have broad benefits to aquaculture
and global food security.
Description Initially we developed the antibody reagents to CD4/CD3 for trout (collaborators at Santiago), and the recombinant viral/bacterial proteins for immunisation and cytokines for our in vitro studies (at Aberdeen), with reciprocal exchange of these reagents. The recombinant cytokines were confirmed to be bioactive prior to use. Analysis of the CD4+ cell distribution within trout tissues was then undertaken. Following on from this experiments were set up to examine in vitro antigen re-stimulation of leucocytes from immunised fish, to assess the cytokine profiles in these cells and the ability of cytokines to modulate the responses, using a model viral and bacteria antigen.

Using the viral model the results showed a big difference in the cytokine expression profile after in vivo stimulation with two viral model antigens of Infectious Pancreatic Necrosis Virus (IPNV), a rhabdoviral disease of salmoinids. VP1, which is a large protein of approx. 94 kDa, stimulated the expression of all the evaluated cytokines (Th1, Th2 and regulatory cytokines) except for IL-2. These results reproduced those found for IFN-? and IL-4 / 13A in an earlier experiment where the immunization conditions were established. A very different response was observed in fish immunized with VP2. In this case, VP2 only increased IL-22, which is a cytokine that is produced by activated T lymphocytes, in a wide range of tissues at sites of inflammation and mediates the protection and regeneration of epithelial tissues. In addition, VP2 decreased IL-2 levels. Following antigen re-stimulation in vitro with VP1 only IFN-? was increased. In contrast, in vitro re-stimulation with VP2 using immune HK-cells induced increased expression of TFG-b, a regulatory cytokine, and decreased expression of IL-22 and IL-10A. We had expected to see a stimulation/ increased expression of cytokines due to further T cell differentiation in vitro, but this did not seem to be the case. Therefore for the cell sorting experiment with this antigen we next took cells from organs just after in vivo immunization.The results of cytokine transcription profile of sorted CD4+ T cells resembled the profile found when total leukocytes of HK and spleen were tested. This is possibly explained by 1) CD4+ T cells are the most abundant cells in the organs after stimulation (T cells should proliferate and differentiate within the lymphoid organs after antigen stimulation) and 2) CD4+ T cells are the cells that mostly producing the cytokines and markers tested.

Using the bacterial antigen VapA from Aeromonas salmonicda, we were unable to detect any clear secondary responses following in vitro re-stimulation. Indeed, introduced VapA commonly reduced responses relative to other treatments. Therefore we tried another antigen, which is a novel antigen called P14G8 from Tetracapsuloides bryosalmona, a myxozoan parasite that causes Proliferative Kidney Disease in farmed trout. This antigen proved to be a better candidate and restimulation induced a clear Th2 type profile, where genes for IL-4/13B1, IL-4/13B2, IL-4Ra1, IL-13Ra2a, IL-6 and IL-7 in particular were all upregulated. These responses were not seen when VapA was added to the P14G8 primed cells, confirming the antigen specificity of the response, and similarly P14G8 did not elicit such responses when added to cells from VapA primed fish. Time course studies revealed that cells from early timings (weeks 1-4) post-immunisation were unable to give a clear response, but at later timings (weeks 6-9) in vitro restimulation gave a consistent increase in cytokine gene expression, potentially relating to expansion of T cell clones over this period in vivo. Cells from fish immunised 8-9 weeks earlier were subsequently used to look at the impact of in vitro cytokine addition at the time of antigen exposure, as in the programe of work.
Exploitation Route Ultimately analysis of protective immune responses post-vaccination may allow more targeted vaccine development for fish, in addition to being an alternative means to assess vaccine efficacy in place of disease challenge.
Sectors Agriculture, Food and Drink

Description A talk at the CAS Institute of Hydrobiology, Wuhan, China, October 2017. 
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 entitled "What do we know about Th1 cells in fish?"
Year(s) Of Engagement Activity 2017
Description A talk at the Symposium "Congreso Uruguayo de Acuicultura. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A talk was given at the above Symposium entitled "Recombinant VP1 and VP2 of infectious pancreatic necrosis virus trigger lymphoid cell changes and induced cytokine transcriptional expression in rainbow trout head kidney".
Year(s) Of Engagement Activity 2018
Description Talk at Asociacion de Estudiantes de Bioquimica, Chile 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A talk was given at the above meeting, entitled "Understanding immunity in salmonids to improve disease resistance in aquaculture.
Year(s) Of Engagement Activity 2018
Description Talk at University of Santiago de Chile 
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 entitled "What do we know about cytokines in fish and what is their relevance to vaccine induced immunity?"
Year(s) Of Engagement Activity 2016