Progress 04/15/16 to 04/14/21
Outputs
Target Audience:
Nothing Reported
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 accounted for in FY20 progress report. FY20 progress report covers FY20 and the partial FY21 year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments accounted for in FY20 statement.
Publications
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Progress 04/15/19 to 04/14/20
Outputs
Target Audience:Our Target Audience includes fish health professionals and the aquaculture industry. The majority of our work is focused the development of tools and resources that address fish health. Changes/Problems:The 2018-2019Govenment shutdown led to significant hiring delays and disruptions for the laboratory during FY19 (Oct 2018 thru Sept 2019). This continued into the next fiscal year (FY20) such that technical assistance was not procured for the project until January 2020. The technician received intial training and then all staff were placed on maximum telework owing to the SARS-CoV-2 induced pandemic. Similar to all laboratories, the pandemic continues to pose problems and time constrainst on research such that the pandemic also affected work conducted by sciencecontractors for the project. 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 at the Conference of Research Workers in Animal Diseases annual meeting and through journal publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
ForObjective A, we produced additional Fc-tagged, soluble proteins for trout MCSFR1, CD3, IFNG as well as Catfish and Tilapia IFNG. These proteins were developed to make corresponding polyclonal antibodies. In addtion, we also produced soluble proteins for trout CD22, CD28, CD83 and CD40/L. Production of tilapia and catfish MCSFR1 and CD3 is ongoing. Hybridomas for trout, catfish and tilapia IFNG were cloned and are being further validated. Secondary confirmation of mAbs against trout CD3 and MCSFR1 are also undergoing validation.ForObjective B, guinea pigs were hyperimmunized withtrout MCSFR1, CD3, IFNG as well as Catfish and Tilapia IFNG and immune sera was collected. Proteins to be affininity purified against target antigen. The second major campaign was initiated for the production of ratmonoclonal antibodies against trout CD28, CD83, CD40/L as well as catfish and tilapia MCSFR1.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Sunyer, J.O., Hansen, J.D., Salinas. I. An update from the Collaborative Immune Reagent Network for Aquacultured Species (CIRNAS). Conference of Research Workers in Animal Diseases. Chicago, ILL. DEC 2019.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Dixon B, Barreda DR, Sunyer JO. Perspective on the Development and Validation of Ab Reagents to Fish Immune Proteins for the Correct Assessment of Immune Function. Frontiers in Immunology. 2018 Dec doi: 10.3389/fimmu.2018.02957.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Hassan KMA, Hansen JD, Herrin BR, Amemiya CT. Generation of Lamprey Monoclonal Antibodies (Lampribodies) Using the Phage Display System. Biomolecules. 2019 Dec doi: 10.3390/biom9120868
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Hansen, J. An update from the Collaborative Immune Reagent Network for Aquacultured Species (CIRNAS). Conference of Research Workers in Animal Diseases. Virtual presentation. DEC 2020.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Salinas I, Zhang YA, Sunyer JO. Mucosal immunoglobulins and B cells of teleost fish. Developmental and Comparative Immunology. 2021 Dec doi:10.1016/j.dci.2011.11.009
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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.
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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.
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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.
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