Progress 04/01/10 to 09/30/15
Outputs
Target Audience: This project presents a broad community plan to begin to systematically address the immunological reagent gap for the US veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses and aquaculture species (concentrating on channel catfish and trout). These will be useful to research scientists for vaccine design and efficacy trials and for those evaluating pathology of important veterinary diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training for students ranging from 3 high school students, 79 undergraduate college students, 24 students in graduate degree programs, and 12 post-doctoral fellows along with technicians and over 20 visiting scientists in project directors' laboratories as well as some training in the labs of other scientists. In addition the website developed provided information on bioassays as well as immune function genes and sequences for scientists around the world. How have the results been disseminated to communities of interest?The results have been disseminated through the website associated with the project: www.vetimm.org that was hosted by the University of Massachusetts Amherst and now by USDA-ARS Beltsville, Maryland since 2015. There were also over 70 scientific presentations at meetings and conferences and smaller seminars as well as over 70 scientific publications in peer-review journals. In addition training of visiting scientists occurred as described under training. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Over the course of this grant which began in 2006had one renewal and a no-cost extension, there was a total of 77 oral presentations given at scientific meetings and over 71 scientific publications. In some cases the publications described new reagent development including gene cloning and generation of monoclonal antibodies and recombinant cytokines and chemokines as well as their use to define cell populations and the function of molecules. These had impact not only on the target agriculture species which contributes to world food supply but in some cases the research on these species served as models for human disease (allergies, infectious diseases) and were also important for research on zoonotic diseases spread from animals to humans.Kingfisher Biotech was the commercial partner of US-VIRN and partially as a result of the interaction with US-VIRN which was in existence at the time of the inception of Kingfisher, an extensive portfolio of reagents for the species targeted by US-VIRN was developed. There were more than 50 cell surface proteins that are markers of leukocyte populations and85 cytokine and chemokine genes cloned and sequenced and deposited in GenBank and many of these were expressed by US-VIRN as recombinant proteins; there was development of approximately 50 monoclonal antibodies, multiplex assays for measuring cytokines for horses and for swine along with other bead-based flow cytometric assays. Other reagents include generating and characterizing recombinant cytokines and chemokines in bioassays developed by the project directors. The reagents have been distributed to all continents worldwide that include companies involved in animal health research and product development, university researchers, scientists at government agencies and research institutes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Baldwin, C.L. and J.C. Telfer. (2014) The bovine model for elucidating the role of ?? T cells in controlling infectious diseases of importance to cattle and humans. Molecular Immunology 66:35-47
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Progress 04/01/14 to 03/31/15
Outputs
Target Audience: Research scientists and clinicians. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two undergraduate and two graduate students received training as part of this project How have the results been disseminated to communities of interest?Results are presented at scientific meetings such as CRWAD in Chicago, IL in December 2014as well as by email from Kingfisher Biotech and on the US-VIRN website. What do you plan to do during the next reporting period to accomplish the goals?Continue to conclude studies as this is a no cost extension year.
Impacts
What was accomplished under these goals?
Additional monoclonal antibodies were produced in species labsand additional products including kits to measure cytokines were released by Kingfisher Biotech.
Publications
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Progress 04/01/13 to 03/31/14
Outputs
Target Audience: This project presents a broad community plan to begin to systematically address the immunological reagent gap for the US veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses and aquaculture species (concentrating on channel catfish and trout). These will be useful to research scientists for vaccine design and efficacy trials and for those evaluating pathology of important veterinary diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduates and graduate students, postdoctoral fellows and technical staff have received training as well as visiting scientists. How have the results been disseminated to communities of interest? Yes through poster presentations at meetings including the 10th International Veterinary Immunology Symposium in Milan, Italy in Aug-Sept 2013 and CRWAD in Chicago in December 2013 in addition to many others as pertains to individual species. Also by generating relevant publications. What do you plan to do during the next reporting period to accomplish the goals? Finish characterizing existing mAbs in the no-cost extension year and for catfish generating new mAbs to recently cloned and expressed proteins including IL-17A and F, IL-15, IL-22, CD4-1 and CD4-2, CD3 gamma and delta and CD115. This is in collaboration with Dr Jeff Hogan, CVM, University of Georgia. Swine reagents being targeted include IL-21, IL-22 and IL-23 p40 with Kingfisher Biotech Inc.
Impacts
What was accomplished under these goals?
A full panel of equine, bovine, swine and chicken cytokines and chemokines genes were cloned and recombinant expressed gene products are available through our commercial arm Kingfisher Biotech. In addition polyclonal antibodies have been developed to some of these and are available for use in ELISA kits and some work for intracellular staining of lymphocytes from Kingfisher Biotech. In addition genes for cell surface molecules were cloned and expressed in mammalian cells or bacteria. These are available at vetimm.org website.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Lee Sung Hyen, Lillehoj Hyun S, Jeong Mi Sun, Del Cacho Emilio, Min Wongi Min, Jeoung Hyeyoung, An Dongjjun, 2014, Development and characterization of mouse monoclonal antibodies reactive with chicken IL1 Beta, Poultry Science
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Lee Sung Hyen, Lillehoj Hyun S, Jeong MiSun, Cacho Emilio D, Min Wongi, Sullivan Yvonne B, Kakach Laura, Labresh Joanna W, 2014, Development and characterization of mouse monoclonal antibodies reactive with chicken TL1A, Veterinary Immunology and Immunopathology, 159:103-109
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Lee SH, Lillehoj HS, Jeong MS, Del Cacho Emilo, Min WG, Sullivan YB, Kakach, L, Labresh JW. 2013. Development and characterization of mouse monoclonal antibodies reactive with chicken TL-1A. Veterinary Immunology and Immunopathology. [Epub ahead of print]
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Min W, Kim WH, Lillehoj EP, Lillehoj HS. 2013. Recent progress in host immunity to avian coccidiosis: IL-17 family cytokines as sentinels of the intestinalmucosa. Dev Comp Immunol. 41(3):418-28.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hamza E, Akdis C, Wagner B, Steinbach F, Marti E. 2013. In vitro induction of functional allergen-specific CD4+CD25high Treg cells in horses affected with insect bite hypersensitivity. Clin. Exp. Allergy, 43: 889-901.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Detournay O, Morrison DA, Wagner B, Zarnegar B, Wattrang E. 2013. Genomic analysis and mRNA expression of equine type I interferon genes. J Interferon Cytokine Res., 33: 746-759.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Silva A, Wagner B, McKenzie HC, Desrochers AM, Furr MO. 2013. An investigation of the role of soluble CD14 in hospitalized, sick horses. Vet. Immunol. Immunopathol., 155: 264-269.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Schnabel CL, Wagner S, Wagner B, Dur�n MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JMV. 2013. Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines. Vet. Immunol. Immunopathol., 156: 1-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Christopher-Hennings J, Araujo KPC, Souza CJH, Fang Y, Lawson S, Nelson E, Lunney JK. 2013. Opportunities for bead based multiplex assays in veterinary diagnostic laboratories. J Vet Diagnostic Investigation. 25: 671-91.
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Progress 04/01/12 to 03/31/13
Outputs
Target Audience: This project presents a broad community plan to begin to systematically address the immunological reagent gap for the US veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses and aquaculture species (concentrating on channel catfish and trout). These will be useful to research scientists for vaccine design and efficacy trials and for those evaluating pathology of important veterinary diseases. To date, Kingfisher Biotech has developed and launched 19 ELISA kits across four species. Regarding the commercialization arm of the grant, Kingfisher Biotech launched an updated website in the past several months and added distributors in Israel and New Zealand. Because of the time lag of grants and publications, the true impact of 500 reagents for veterinary research will not be realized for several years. There are already 17 scientific papers citing the use of Kingfisher Biotech products in veterinary research and many more in progress. Because of scientist requests, products now cover 13 species. The greatest hurdle we now face is how to effectively get the word out about all the products that are now available. For trout, both cells and supernatant from VIRN-made immune reagents have been requested by, and shipped to, 6 labs in the United States, 2 labs in South America, and 5 labs in the European Union. For chicken, 23 genes in plasmids, 13 of recombinant proteins, 94 mAbs, and 17 pAbs were sent to 15 institutes and universities including Semmelweis Univ in Hungary, University of Zaragoza in Spain, and FDA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduates and graduate students, postdoctoral fellows and technical staff have received training. How have the results been disseminated to communities of interest? Publications and presentations at scientific meetings. Examples of presentations during 2012 are here: Baldwin CL, Bromage E, Lunney J, Lillehoj H, LaBresh J, Hansen J, Bengten E, Wilson M, Wagner B. US Veterinary Immune Reagent Network. USDA-NIFA Project directors Workshop, Chicago, IL 12/2012. Tuggle CK, Rogel-Gaillard C, Loveland J, Uenishi H, Dawson H, Lunney J, Sang Y, Zhao S, Giuffra E, Botti S, Reecy J, Harrow J, Freeman T, Archibald AL, Murtaugh M, Hume D, Morozumi T, Shinkai H, Bed’hom B, Blecha F, Zhang G, Mann K, Zhang J Chen C, Hu Z, Cheng R, Huang T, Rodriguez Y, Anselmo A, Badaoui B, Schwartz J, Kapetanovic R, Dario Beraldi D, and Immune Response Annotation Group (IRAG) and the Swine Genome Sequencing Consortium. (2012) Structural and functional annotation of immunity-related genes in the pig genome. Plant and Animal Genome Conference 2012, January 14-18, 2012, San Diego, CA. PAG P3622. http://pag.confex.com/pag/xx/webprogram/Paper3622.html Wagner B. – invited speaker – Multiplex technology – A new sensitive method to measure soluble analytes. Colorado State University. Orthopaedic Research Center, 4/23/2012. Wagner B. – invited speaker – Immune mechanisms that substitute T-helper cell responses in neonates and young foals. Havemeyer Rhodococcus equi Workshop, Deauville France, July 9-12, 2012. Wagner B. – invited speaker – Cellular immune responses to equine herpesvirus type 1 in foals and adult horses. Annual Meeting American College of Veterinary Pathologists. Seattle, WA, Dec. 1-5, 2012. Wilson, M., What we know and what we don't know about channel catfish B and T cells. College of Veterinary Medicine University of Georgia, Athens Georgia, March 26, 2012. (Oral Presentation) Wilson, M., What we know and what we don't know about channel catfish B and T cells; 3rd North American Comparative Immunology Workshop, University of Rochester, Rochester, New York, June 5-8, 2012 (Plenary lecture) What do you plan to do during the next reporting period to accomplish the goals? Work hard pursuing the directions we have established.
Impacts
What was accomplished under these goals?
A full panel of equine, bovine, swine and chicken cytokines and chemokines genes were cloned and recombinant expressed gene products are available through our commercial arm Kingfisher Biotech. In addition polyclonal antibodies have been developed to some of these and are available for use in ELISA kits and some work for intracellular staining of lymphocytes from Kingfisher Biotech. In addition genes for cell surface molecules were cloned and expressed in mammalian cells or bacteria. For cell surface molecules and cytokines and chemokines a total of 16 mAbs for horse molecules, 6 mAbs for catfish molecules, 6 mAbs for cattle molecules, more than 20 mAbs to chicken molecules, 12 mAbs to swine molecules and 6 mAbs to trout molecules have been produced and characterized.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2013
Citation:
Hamza E, Akdis C, Wagner B, Steinbach F, Marti E. In vitro induction of functional allergen-specific CD4+CD25high Treg cells in horses affected with insect bite hypersensitivity. Clin Exp Allergy. In press.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2013
Citation:
Keggan A, Freer H, Rollins A, Wagner B. Production of seven monoclonal equine immunoglobulins isotyped by multiplex analysis. Vet Immunol Immunopathol. In press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Wagner B, Hillegas J, Kabithe E, Boyd P, Zarlenga D, Zagorski B, Dawson H, Lunney, JK. 2013. Development and characterization of a monoclonal antibody to porcine interleukin 4 receptor alpha (CD124). Vet Immunol Immunopathol. In revision.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Butler JE, Wertz N, Sun XZ, Lunney JK, Muyldermans S. 2013. Resolution of an immunodiagnostic dilemma: Heavy chain chimeric antibodies. Molec Immunol. 53: 140148.
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Progress 04/01/11 to 03/31/12
Outputs
OUTPUTS: This report includes progress by both duty labs and species labs for the second year of the renewed US-VIRN grant. It is accompanied by a comprehensive list of publications. We also have made many presentations regarding US-VIRN and its progress at scientific meetings and other science-oriented venues which are not in the report. Additional information on progress and plans and access to written and oral presentations may be found at www.vetimm.org, our US-VIRN website. PARTICIPANTS: Project Directors and Institutions: C.Baldwin, E. Bromage, J. Lunney, H. Lillehoj, J. LaBresh, J. Hansen, E. Bengten, and M. Wilson, B. Wagner. University of Massachusetts, Amherst and Dartmouth, MA, USDA-ARS, Beltsville, MD, Kingfisher Biotech, Minneapolis, MN, Western Fisheriers Res Ctr USGS, Seattle, WA, University of Mississippi Medical Center, Jackson, MS, Cornell University, Ithaca, NY TARGET AUDIENCES: This project presents a broad community plan to begin to systematically address the immunological reagent gap for the US veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses and aquaculture species (concentrating on channel catfish and trout). These will be useful to research scientists for vaccine design and efficacy trials and for those evaluating pathology of important veterinary diseases. To date, Kingfisher Biotech has developed and launched 19 ELISA kits across four species. Regarding the commercialization arm of the grant, Kingfisher Biotech launched an updated website in the past several months and added distributors in Israel and New Zealand. Because of the time lag of grants and publications, the true impact of 500 reagents for veterinary research will not be realized for several years. There are already 17 scientific papers citing the use of Kingfisher Biotech products in veterinary research and many more in progress. Because of scientist requests, products now cover 13 species. The greatest hurdle we now face is how to effectively get the word out about all the products that are now available. For trout, both cells and supernatant from VIRN-made immune reagents have been requested by, and shipped to, 6 labs in the United States, 2 labs in South America, and 5 labs in the European Union. For chicken, 23 genes in plasmids, 13 of recombinant proteins, 94 mAbs, and 17 pAbs were sent to 15 institutes and universities including Semmelweis Univ in Hungary, University of Zaragoza in Spain, and FDA. PROJECT MODIFICATIONS: During the PI meeting in Jan 2012 it was decided that efforts to produce anti-T cell receptor alpha and beta TCR mAb at Cornell should be discontinued due to the poor results from at least 5 different trials, using each porcine gene expressed with mammalian system either separately or together (rPoTRAC/TRBC).
Impacts
For swine we are developing new multiplex assays and completed development of one for CCL2. For swine, 11 cytokines, 8 chemokines and 3 interferons have been expressed and are sold by Kingfisher Biotech. At Cornell, swine NCR2, NCR3 and CD19 have been expressed and NCR2 used for mouse immunization. At UMass monoclonal antibodies (mAbs) to swine proteins expressed at UMass in mammalian cells or in yeast by Kingfisher were made as follows, 1 to IFN-alpha, 4 for IFN-beta, 18 for IL-13, 2 for IL-17A and 4 for IL-6. For cattle, several mAb clones and subclones that react with mammalian-expressed bovine IL-6 have been developed along with 4 mAbs against mammalian-expressed bovine IL-17F. For horse, mAb production is in progress or finished for equine CD25 and TCR alpha. Mab characterization for equine IL-5 and CCL11 is ongoing. For chicken, gene cloning for IL5R, IL7, IL7R, IL10R-beta, IL12p35, IL13, IL17R, and IL21R was accomplished and 6 chicken IL7, IL13, IL21, IFN-alpha, CXCL14 and lymphotactin are being expressed at Kingfisher while IL1R, IL5R, IL7R, IL10R, and IL21R are being expressed at Cornell in mammalian cells with chicken IL-7R purified for mAb production. Proteins expressed in E. coli and bioassayed include chicken IL5R, IL7, IL7R, IL12p35, IL13, and IL21R in Lillehoj lab. Chicken IL6, IL17F, IL-22 and turkey IL1-beta and turkey IL8 have been expressed by Kingfisher and evaluated in bioassays by Lillehoj lab with L17F, IL1beta and IL8 shown to be active. MAbs have been developed against E.coli-expressed chicken cytokines (IL2, IL4, IL6, and IL10) in the Lillehoj lab. For catfish, a new anti-IgD mAb was developed and characterized, TCR-alpha recombinant protein produced and final hybridoma screenings ongoing. New targets for hybridomas are TCR-gamma and CD2 and CSFR1. For trout, several new mAbs using eukaryotic expression systems and peptide-based approaches includes rainbow trout IgD, both secreted and membrane-associated forms and mAbs specific for trout CD3-epsilon produced and characterized. Finally, the trout responded to out of network requests to generated mAbs specific for pacific herring and flounder and the anti-herring mAbs used as part of the herring restoration efforts on the west coast. MAb against IL1-B are in the testing phase with 16 clones for pairing in an ELISA or luminex assay. Recombinant proteins were made against IL6, IFN-gamma, complement component 3d, and IgT. Kingfisher Biotech continues to progress on expressing proteins for veterinary species, including bovine, equine, catfish, chicken/turkey and swine. Over the past year, we have cloned, expressed and purified over 12 new proteins, including chicken IFN alpha, swine IL-1 receptor antagonist, catfish IL-8, and bovine IL-10 and APRIL. We tried several new strategies this year, the synthetic gene design and implementation into our process successfully resulting in bovine IL-10 protein being available. We also recently cloned and expressed proteins with 2 different chains, notably the IL-12 protein, which has both an A and a B chain. Generation and affinity purification of polyclonal antibodies was done and these have been used for ELISA kits.
Publications
- Lee, S.H., Lillehoj, H.S., Jang, S.I., Baldwin, C., Tompkins, D., Wagner, B., Parcells, M., Del Cacho, E., Hong, Y.H., Min, W., Lillehoj, E.P. 2011. Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor alpha chain (CD25). Vet Immunol Immunopathol. 144:396-404.
- Lee, J.J., Yoo, J., Koh, P.O., Kim, Y.H., Chang, H. H., , Choe, N.H., Lillehoj, H. S.,, Min, W. 2011. Molecular identification of duck and quail common cytokine receptor gamma chain genes. Vet Immunol Immunopathol. 140:159-65.
- Lee, S.H., Lillehoj, H.S., Jang, S.I., Lee, K.W., Baldwin, C., Tompkins, D., Wagner, B., Del Cacho, E., Lillehoj, E.P., Hong, Y.H. 2012. Development and characterization of mouse monoclonal antibodies reactive with chicken CD83. Veterinary Immunology and Immunopathology, 145: 527-533.
- Wimer CL, Damiani A, Osterrieder N, Wagner B. 2011. Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression. Vet. Immunol. Immunopathol. 140: 266-274.
- Wagner B, Wimer C, Freer H, Osterrieder N, Erb HN. 2011. Infection of peripheral blood mononuclear cells with neuropathogenic equine herpesvirus type-1 strain Ab4 reveals intact interferon induction and induces suppression of anti-inflammatory interleukin-10 responses in comparison to other viral strains. Vet. Immunol. Immunopathol, 143: 116-124.
- Robbin MG, Wagner B, Noronha LE, Antczak DF, deMestre AM. 2011. Subpopulations of equine blood lymphocytes expressing regulatory T cell markers. Vet. Immunol. Immunopathol. 140: 90-101.
- Goodman LB, Wimer C, Dubovi E.J, Gold C, Wagner B. 2012. Immunological correlates of vaccination and infection for equine herpesvirus type 1. Clin. Vacc. Immunol, 19: 235-241.
- Wattrang E, Palm AK, Wagner B. 2012. Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 146: 113-124.
- Wagner B, Hillegas J, Babasyan S. 2012. Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses. Vet. Immunol. Immunopathol. 146: 125-134.
- Noronha LE, Harman RM, Wagner B, Antczak DF. 2012. Generation and characterization of monoclonal antibodies to equine CD16. Vet. Immunol. Immunopathol. 146: 135-142.
- Noronha LE, Harman RM, Wagner B, Antczak DF. 2012. Generation and characterization of monoclonal antibodies to equine NKp46. Vet. Immunol. Immunopathol. 147: 60-68.
- Jeong, J., Kim, W.H., Yoo, J., Lee, C., Kim, S., Cho, J.H., Jang, H.K., Kim, D.W., Lillehoj, H.S., Min, W. 2012. Identification and comparative expression analysis of interleukin 2/15 receptor beta chain in chickens infected with E. tenella. PLosOne (5):e37704.
- Ramirez-Gomez F, Greene W, Rego K, Hansen JD, Costa G, Kataria P, Bromage ES. 2012. Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism. J Immunol. 188:1341-9.
- Boardman T, Warner C, Ramirez-Gomez F, Matrisciano J, Bromage E. 2012. Characterization of an anti-rainbow trout (Oncorhynchus mykiss) CD3 epsilon monoclonal antibody. Vet Immunol Immunopathol. 145:511-5.
- Purcell MK, Bromage ES, Silva J, Hansen JD, Badil SM, Woodson JC, Hershberger PK. 2012. Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii). Fish Shellfish Immunol. 33:552-8.
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Progress 04/01/10 to 03/31/11
Outputs
OUTPUTS: This report includes progress by both duty labs and species labs for the first year of the renewed US-VIRN grant and plans for the second year. It is accompanied by a comprehensive list of publications (26 total for 2010-2011). We also have made many presentations regarding US-VIRN and its progress at scientific meetings and other science-oriented venues which are not in the report (20 total for 2010-2011). Additional information on progress and plans and access to written and oral presentations may be found at www.vetimm.org, our US-VIRN website which is regularly updated. Over the summer 2010 an extensive reworking of the website was undertaken after consultation with the PDs and members of the veterinary immunology community. The goal was to make it more accessible to individuals attempting to find the Network information as well as to update overall. The resultant changes have dramatically increased the profile of the site through better web search engine hits. PARTICIPANTS: Project Directors and Institutions: C.Baldwin, E. Bromage, J. Lunney, H. Lillehoj, J. LaBresh, J. Hansen, E. Bengten, and M. Wilson, B. Wagner. University of Massachusetts, Amherst and Dartmouth, MA, USDA-ARS, Beltsville, MD, Kingfisher Biotech, Minneapolis, MN, Western Fisheriers Res Ctr USGS, Seattle, WA, University of Mississippi Medical Center, Jackson, MS, Cornell University, Ithaca, NY TARGET AUDIENCES: This project presents a broad community plan to begin to systematically address the immunological reagent gap for the US veterinary immunology research community including for the following groups: ruminants (concentrating on cattle), swine, poultry (primarily chickens with some evaluation of reagents on turkey cells), horses and aquaculture species (concentrating on channel catfish and trout). These will be useful to research scientists for vaccine design and efficacy trials and for those evaluating pathology of important veterinary diseases. PROJECT MODIFICATIONS: Several new approaches for monoclonal antibody production have been integrated into the project and include the development of two new expression vectors.
Impacts
Kingfisher Biotech developed new strategies to clone, express or purify chicken IL-17A and D, swine IFN alpha, swine IL-15, equine IL-15 and expressed chicken IL-6, swine IL-6, swine IL-17F, and turkey IL-8. At Cornell nine proteins were expressed including cattle IL-23R, chicken CXCR4, equine TCR alpha, TCR beta, IL-1 beta, IL-13, IL-15, GM-CSF and TLR2. Using the IL-4 expression system, equine TLR2 was secreted and is currently being tested for mAb production. New genes for expression include swine and horse NK-receptors (NCR2 and NCR3) and CD19. mAb development is/was ongoing for swine TCR alpha and beta and five horse proteins and mAbs to equine CCL2, CCL3, CCL5 and IL-17A were successfully tested in various assays using the corresponding native cytokines. Lunney lab completed bioactivity assays for swine chemokines CCL3, CCL4, CCL5 and screened a fusion for swine TCR and immunizations for swine IL-6 and IFN-beta mAb and IL-17A/CCL3L1 are underway and fusions expected soon. At UMass, bioassays for bovine and equine IFN gamma displayed bioactivity in conjunction with TNF alpha in our standard protocol. An alternative bioassay for IL-13 that relies on measuring upregulation of MHC Class II expression was developed. MDBK-t2 cells and a CAT ELISA system were used to determine the bioactivity of yeast- and mammalian-expressed type I interferons for mammalian and yeast-expressed bovine, equine and swine and all were bioactive. Mouse immunizations with Kingfisher yeast-expressed protein: bovine IL-6/IL-13, swine IL-6/IFN beta; swine IL-17A/CCL3L1 are underway. For trout, efforts focused on mAbs to trout B-cell (IgD, sIgM, IgT, CD79A) and T-cell (CD3, CD4, CTLA4 and TcR) surface receptors and accessory molecules. They utilize three approaches for immunizing mice; prokaryotically expressed HIS-tagged fusion proteins (E.coli), eukaryotically expressed FC-tagged fusion proteins (HEK293 cells) and a peptide-based approach. A peptide approach was used for IgD, secIgM, IgT, CD3 and CD4, and successfully produced anti-IgD and anti-secIgM mAbs that have been validated. The trout group received requests for anti-IgM mAbs for Pacific herring, yellowtail flounder and yellow perch due to specific disease issues. 17 fusions were performed, resulting in 4 products (trout IgD, trout secIgM, Herring IgM, Flounder IgM). For catfish, efforts have been focused on developing mAbs to IgD, IgL lambda, beta 2microglobulin, TcR gamma delta and CD3 gamma delta. Catfish recombinant IgD, IgL lambda, beta 2microglobulin, and CD3 gamma delta protein were produced in E. coli. Peptides based on IgD, CD8 and CD4 were made. For poultry, eleven chicken cDNA homologs were cloned(CXCL4, GM-CSF, IFN-gamma, IL12p40, IL17A, IL17D, IL21, IL22, LITAF, Lymphotactin, and TNFSF) and sent to Kingfisher. Protein expression and bioassay development for chicken IL4, IL12, GM-CSF, IFN-gamma, and TNFSF, and turkey IGF1 occurred; turkey IGF2 was evaluated for bioactivity. CD25 and CD80 were used for mAbs and IL-18 expressed at ARS was shown to be biologically active. Four hybridomas have been developed against E.coli-expressed IL-17A, IL17D, IFN-gamma, and TL1A.
Publications
- Wagner B, Stokol T and Ainsworth DM. 2010. Induction of interleukin-4 production in neonatal IgE+ cells after crosslinking of maternal IgE. Dev. Comp. Immunol. 34: 436-444.
- Hudgens E, Tompkins D, Boyd P, Lunney JK, Horohov D, Baldwin CL. 2011. Expressed gene sequence and bioactivity of the IFNγ-response chemokine CXCL9 of cattle, horses and swine. Vet. Immunol. Immunopathol., In press.
- Laing, KJ., and Hansen, J.D. 2011. Fish T-cell: advances through genomics (Review). Developmental and Comparative Immunology, In press.
- Lee, SH, Lillehoj, HS, Park, MS, Baldwin, C, Tompkins, D, Wagner, B, Del Cacho, E, Babu, U, Min, W. 2011. Development and characterization of mouse monoclonal antibodies reactive with chicken CD80. Comp Immunol Microbiol, In press.
- Quiniou S, Sahoo M, Edholm ES, Bengten E, Wilson M. Channel catfish CD8a and CD8b co-receptors: characterization, expression and polymorphism. 2011. Fish and Shellfish Immunology, Jan 25. [Epub ahead of print] PMID: 21272650.
- Wagner B, Burton A and Ainsworth DM. 2010. Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals. Vet. Res. 41: 47-61.
- Edholm ES, Wilson M, Bengten E. 2011. Immunoglobulin light (IgL) chains in ectothermic vertebrates (review). Developmental and Comparative Immunology, Jan 21. [Epub ahead of print] PMID: 21256861
- Edholm ES, Bengten E, Wilson M. 2011. Insights unto the function of IgD (review). Developmental and Comparative Immunology, In press.
- Entrican G, Lunney JK. 2011. Veterinary Immunology Committee Toolkit Workshop 2011: Progress and Plans. Vet. Immunol. Immunopathol., In press.
- Robbin MG, Wagner B, Noronha LE, Antczak DF,de Mestre AM. Subpopulations of equine blood lymphocytes expressing regulatory T cell markers. 2011. Vet. Immunol. Immunopathol.140:90-101
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