Source: GEOLOGICAL SURVEY, UNITED STATES DEPT OF submitted to
COLLABORATIVE IMMUNE REAGENT NETWORK FOR AQUACULTURED SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1009003
Grant No.
2016-67015-24901
Project No.
WN.W-2015-07477
Proposal No.
2015-07477
Multistate No.
(N/A)
Program Code
A1223
Project Start Date
Apr 15, 2016
Project End Date
Apr 14, 2021
Grant Year
2016
Project Director
Hansen, J. D.
Recipient Organization
GEOLOGICAL SURVEY, UNITED STATES DEPT OF
6505 NE 65TH ST
SEATTLE,WA 98115
Performing Department
(N/A)
Non Technical Summary
Half of the seafood eaten worldwide is farm raised and therefore it is not surprising that aquaculture represents a rapidly growing industry. Aquaculture also supports the national and commercial sport fisheries. In general, fish are a low-fat source of high quality protein and are the main source of omega-3 fatty acids--these fatty acids prevent and aid in the treatment of human disorders including high blood pressure, heart disease and cancer. Therefore it is easy to appreciate that the sustainable production of healthy, robust finfish is of paramount importance for human health. Unfortunately, disease in the aquaculture sector hinders production. Disease prevention requires continuous monitoring of fish health status as well as the implementation of prophylactic measures for limiting disease. Progress in the area of disease prevention is limited due to the lack of quality veterinary and research-grade immune-related tools for evaluating fish health and correlates of vaccination. Therefore the ultimate goal of this project is to develop and provide immunological tools and assays to the aquaculture community to advance fish health. The main benefit of this research will be to enhance sustainable aquaculture in the US.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31108101090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
Aquacultured fish represent a critical food resource and efficient means for providing protein forhuman consumption. However, disease limits the sustainability and production of this industry. Amajor factor restricting advancement of basic and applied research for fish health is the lack ofimmunological reagents. These reagents are required for evaluating shifts in immunity duringinfection and vaccination against pathogens that limit the full potential of aquaculture. Thisresearch project addresses the need for the development of immune reagents for aquaculture fishto overcome obstacles that limit advances in farmed fish health. In particular, four fish species:rainbow trout, Atlantic salmon, channel catfish and Nile tilapia will be addressed. Antibody panelsand immune assays will be developed to assess the contribution of leukocyte subsets and effectormolecules that cannot be currently measured during disease and vaccination due to lack ofcorresponding reagents. Specific focus areas include the development of reagents for mucosalimmunity and vaccinology. A major objective of this proposal is to establish a strong collaborativenetwork between research professionals for improving the health, safety and production of aquacultured fish. By doing so, the projectwill advance the development of collaborative research projects for diseases impacting production.The achievement of these goals will be enhanced by synergistic efforts of national and internationalcollaborators that are involved in the project. Overall, this proposal will generate a vast knowledgebase, will assist researchers in the aquaculture community to determine fish health status, improveanimal welfare and implement targeted immune interventions for disease.
Project Methods
Recombinant proteins will be produced using eukaryotic and prokaryotic cells. These proteins will be used to immunize mice and rabbits for the production of monoclonal and polyclonal antibodies specfic for proteins expressed on or within finfish leukocytes.

Progress 04/15/18 to 04/14/19

Outputs
Target Audience:Our main target audience includes fish health researchers and the aquaculture sector. The majority of our work is focused on the development and verification of new immune reagents for fish health. Changes/Problems:We had techinical issues with high throughputsecondary screening that were resolved through consultation with other members of NIFA funded reagent grants. In addition, emphasis will be placed on the development of immune targets that are expressed on the cell surface of leukocytes as our high throughput approach is not suitable for cytoplasmic/secreted proteins (e.g. cytokines). What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results were presented at the Annual NIFA PD meeting in Chicago, ILL December 2018 and at the ISDCI Congress in Sante Fe, NM (June 2018) What do you plan to do during the next reporting period to accomplish the goals?Main goals. For Objective A, we will develop additional constructs for trout, catfish and tilapia immune targets. Primary emphasis will be on markers exprssed on the cell surface of leukocytes. For Objective B will will confirm hybridoma clones, assess reactivity/specificity of pAbs and move forward with the next high throughput immunization and screening campaign including trout CD22, CD28, CDS83 and catfish/tilapia CD3.

Impacts
What was accomplished under these goals? ForObjective A, we generated additional expression costructs for rainbow trout CD22, CD83, CD40/L, CD86, CD28. In additon, plasmids encoding tilapia and catfish CD3 and MCSFR1 we produced. Soluble, murine Fc tagged proteins for CD22, CD28, CD40, CD3 and MCSFR1 were produced using HEK293 suspension cells. As forObjective B, we continued our screening of the high throughput immunizaiton trial involving trout CD3gd, MCSFR1 using protein on bead screening and the use of primary leukocytes. For trout, catfish and tilapia IFNG, we expressed those targets on the surface of NIH3T3 cells for 2° screeing. Postive hybridomaswere cloned and are being characterized. In addition, proteins were used for the production of polyclonal antibodies, the reactivity of which has yet to be tested.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: J.D. Hansen, O.J. Sunyer and Salinas, I. Collaborative immune reagent network for aquaculture species. NIFA PD Meeting. Chicago, ILL December, 2018.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: J.D. Hansen, F. Takizawa, I Salinas, and Sunyer, O.J. Collaborative immune reagent network for aquaculture species-an update. International Society of Comparative and Developmental Immunology Congress. Santa Fe, NM June 2018.


Progress 04/15/17 to 04/14/18

Outputs
Target Audience:Primary target audience includes fish health researchers. This comprises but is not limited to fish disease research and vaccine development but also for researchers studying basic aspects of the fish immune system. This incorporates researchers using rainbow trout, salmon, catfish and tilapia as their research model(s). The majority of our efforts are focused on the development of new immune reagents and tools for fish health. Distribution of tools developed within CIRNAS will be distributed through our recently developed website (http://biology.unm.edu/cirnas/) that is maintained at the University of New Mexico. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results were disseminated to at the Annual NIFA PD Meeting in Chicago, Ill. December 2017. What do you plan to do during the next reporting period to accomplish the goals?For Objective A, we will continue to use the CHO-S expression system for the production of soluble proteins for rainbow trout, salmon, tilapia and catfish. Research community has interest in T-cell reagents for catfish and tilapia. To achieve these needs, catfish and tilapia CD3GD will be produced. The majority of effort will be focused on verifying putative positive clones obtained through the high throughput immunization and screening protocol for trout CD3GD, MCSFR1 and IFNG as well for catfish and tilapia IFNG.

Impacts
What was accomplished under these goals? In Objective A, we used eukaryotic cells (CHO-S) to express soluble forms of target protein with a murine Fc tag for purification. We obtained mg amounts of soluble protein for rainbow trout CD3GD, MCSFR1 and IFNG. In addition we obtained mg amounts of tilapia and catfish IFNG using the CHO-S expression system. For Objective B, we initiated a high throughput immunization and screening system in collaboration with the Fred Hutchinson Cancer Researc Center for the development of monoclonal antibodies. Mice were immunized with proteins from objective A and initial "protein on bead" screening was conducted by flow cytometry. Putative positive hybridomas were found for all target proteins.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Salinas, I., Sunyer, O.J., and J.D. Hansen. Collaborative immune reagent network for aquacultured species (CIRNAS). NIFA PD Annual Meeting. Chicago, Ill. December 2017.


Progress 04/15/16 to 04/14/17

Outputs
Target Audience:Primary target audience includes fish health researchers. This comprises but is not limited to fish disease research and vaccine development but also for researchers studying basic aspects of the fish immune system. This incorporates researchers using rainbow trout, salmon, catfish and tilapia as their research model(s). The majority of our efforts are focused on the development of new immune reagents and tools for fish health. Distribution of tools developed within CIRNAS will be distributed through our recently developed website (http://biology.unm.edu/cirnas/) that is maintained at the University of New Mexico. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Posters and oral presentaions were given three different national meetings including 1) 2nd Annual Fish and Shellfish Conference (Portland, ME 2016), 2). NIFA Annual PD Meeting (Chicago, Ill 2016) and 3) Aquaculture America 2017. What do you plan to do during the next reporting period to accomplish the goals?Our individual laboratories and high throughput contractor will produce panels of monoclonal antibodies against CD3GD, CD56, IFNG and TCR for target species. Co-PIs will assess specificity of the reagents including flow cytometric analyses, intracellular staining, bioactivity, protein biochemistry and finally LCMS verification as possible. Our website will provide updates for the target audience. For objective A, we will continue to produce target proteins for trout, salmon, catifsh and tilapia. These protein targets include but are limited to CD28, CLTA4, TCF and MCSFR. For objective B, the participants will focuson screening potential reagents against CD3, IFNG and CD56.

Impacts
What was accomplished under these goals? InObjective A, we will use both prokaryotic and eurkaryotic approaches for expressing recombinant fish proteins. For some protein targets, peptides will be synthesized and conjugated to keyhole limpet hemocyanin (KLH) for immunization. For prokaryotic expression, we will express specific proteins that are fused to glutathione serum transferase (GST). Proteins will be expressed in E.coli, purified and refolded.Two different expression systems will be used for eukaryotic expression. Soluble proteins will be expressed as murine Fc fusion proteins using CHO-S cells under transient transfection conditions. In addition, stable cell lines will be derived that display the extracellular domains of target proteins on the surface of rodent cell lines. In Objective B, mice or rats will be injected with fusion-tagged proteins (from objective A) for the production of hybridoma cell lines that secrete antigen-specific monoclonal antibodies. We will employ both traditional methods (one target at a time) as well as recently derived, high throughput methodologies for producing monoclonal antibodies. Specific responses will be screened by ELISA, western blot, immunoprecipitation, flow cytometry and when applicable--by LCMS of immunoprecipitated proteins. In addition, polyclonal antibodies will be produced against cytokine and chemokine-based targets for the development of ELISAs and ELISPOT assays. Reagents developed within this award will be distributed through http://biology.unm.edu/cirnas/. Accomplishments:For Objective A, project participants have cloned specific targets (e.g. CD3GD, IFNG1, CD56)for all species (trout, salmon, catfish and tilapia) into eukaryotic expression vectors for the production of recombinant proteins. Proteins have been expressed using HEK293 and CHO-S suspension cell systems. Production of additonal protein targets is ongoing. Production of approximately 20% of the protein targets has been achieved. For Objective B-"not started yet". Overall impacts:none to date; project is ongoing and concludes April, 2019.

Publications

  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2016 Citation: Salinas, I., Sunyer, O.J., and J.D. Hansen. CIRNAS: Collaborative immune reagent network for aquacultured species. 2nd International Conference of Fish and Shellfish Immunology. Portland, ME. June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Salinas, I., Sunyer, O.J., and J.D. Hansen. Collaborative immune reagent network for aquacultured species (CIRNAS). NIFA PD Annual Meeting. Chicago, Ill. December 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sepahi, A., Salinas, I., Sunyer, O.J. and J.D. Hansen. CIRNAS: Collaborative immune reagent network for aquacultured species. World Aquaculture Society-Aquaculture America 2017. San Antonio, TX. February 2017.