Progress 02/15/15 to 02/14/20
Outputs
Target Audience:Our major target audience is swine immunologists. This includes scientists working in swine disease analyses and vaccine development, as well as those involved in developing novel biotherapeutics and nutritional interventions. It includes scientists using the pig as a biomedical model, e.g., for development, obesity, cardiovascular, allo- and xeno-transplantation, and vaccine research. Our efforts are targeted at developing new immune reagents and assays for all of these researchers. Changes/Problems:changes noted in report. Grant now completed. What opportunities for training and professional development has the project provided?A graduate student and technician have been trained to perform ELISA and flow cytometry assays for screening of pig CD1d specific mouse serum samples and for screening of IFNg mAb at the Ohio State University. Once HBCU Pathways student and two international Visiting Scientists (a collaborator from the Korean Rural Development Administration, National Institute of Animal Science, Republic of Korea and a PhD student from China supported by Yangzhou University International Academic Exchange Fund YZUIAEF201901005) were trained at BARC in mAb screening techniques. A graduate student was trained at the Univ. of Bristol in performing immunohistochemistry tests of mAbs. How have the results been disseminated to communities of interest?Posters were presented at relevant meetings: yearly at the Conference of Research Workers in Animal Diseases; yearly at the British Society for Immunology; once at the American Association of Immunologists meeting; once at the 14th International Xenotransplantation Association Congress; once at the 6th Swine in Biomedical Research Conference; once at the European Veterinary Immunology Workshop, and once at the International Veterinary Immunology Symposium. Data is updated on the USDA Porcine Translational Research Database (http://tinyurl.com/hxxq3ur). ). Efforts were discussed with and coordinated internationally through the International Union of Immunological Societies Veterinary Immunology Committee Toolkit Committee and in the last year coordinated with the newly formed UK Immunological Toolbox (https://www.immunologicaltoolbox.co.uk/). Several manuscripts have been published; others are in preparation for submission to peer review journals. ? What do you plan to do during the next reporting period to accomplish the goals?final report - grant is completed.
Impacts
What was accomplished under these goals?
Objective 1: Clone and express swine immune cytokines and chemokines. The team developed priority lists of porcine immune proteins.We worked with our commercial partner, Kingfisher Biotech, Inc., where most of the markers were cloned and the proteins expressed using their yeast Pichia pastoris system and then purified. Proteins were provided to the team as well as made as commercial products available on their website https://www.kingfisherbiotech.com/. At Ohio State a major effort was undertaken to express CD1d. Computer designed peptides (coupled to KLH) were used as immunogens in CD1d knockout (KO) Balb/c mice; mouse sera were tested on conventional and CD1d knockout pig peripheral blood mononuclear cells (PBMCs). While some reactivity was seen via ELISA against the synthetic peptides, the sera reacted with PBMCs from conventional and CD1d knockout PBMCs. Next, the porcine CD1d sequence was successfully cloned in frame with EYFPand transient expression tested in Huh7 liver cells and swine testicular cells but failed to produce an eYFP signal in cells or in western blots. Third, the NIH tetramer facility at Emory Univ. agreed to synthesize porcine CD1d [they had generated human and mouse CD1d tetramers]. They used the poCD1d sequence expressed in lenti-virus system; but no expression was reported. Multiple efforts for Ohio State to obtain an MTAfor the lenti expression system were unsuccessful. This effort was terminated. Objective 2: Prepare panels of mAb reactive with swine targets. The immune proteins produced during this grant included IL-6, IL-13, IL-17A, CXCL10 CX3CL1, IFNb, IFNg, IFNw1, and IFNw5. Panels of mAbs were produced for each protein and characterized, and assays established for certain targets. At the start of this grant companies were contacted to perform immunizations, fusions, hybridoma cloning and immunoglobulin (Ig) purifications. However, one company was not productive; it provided a limited number (1-3) of cloned hybridomas for IFNw1 and IFNw5 targets. Further efforts with this company were discontinued. Results from the 2nd company were productive (9-12 mAbs recognizing 3 to 8 epitopes/target protein). Only one protein, CX3CL1, had poorer results: 3 hybridomaseven after 3 fusions. The top 10-15 hybridomas for each target were cloned, grown, mg quantities purified and provided to BARC. Once characterized will be available to the community as commercial products. At BARC purified mAbs were biotinylated and ELISAs used to identify epitope reactivity, based on cross inhibition assays and on analyses of mAb cross reactivity on orthologous proteins from other species (supplied by Kingfisher Biotech Inc.). Epitope reactivity was completed for 7 proteins with only IL-6 having few, just 3, epitopes. By targeting unique epitopes fewer mAb pairs needed to be tested for development of sandwich ELISAs for quantitating these immune markers; however, if there are too few mAbs (CX3CL1) or epitopes (IL-6) future uses may be restricted. At the University of Bristol, the 3 anti-CX3CL1 mAbs were tested for gut staining. Strong villi staining was observed for one antibody and follicular staining was observed for a different antibody. One IFNw5 mAb was visualized using immunofluorescence on nasal tissues and demonstrated high levels of staining of influenza mediated lung lesions. The UK Univ. Bristol researchers selected cell surface chemokine receptors (CCR3, CCR9 and CCR10) as their target markers. They worked with the AbD Serotec HUCAL phage display system with the extracellular loop 2 (ECL2) sequence as bait. The HUCAL system identified three, two and eight bivalent Fab fragments [F(ab)2], originating from phage display searches against synthetic, chemically constrained peptides corresponding to ECL2 of CCR3, CCR9 and CCR10, respectively. All CCR3 F(ab)2s and one CCR9 F(ab)2 stain PBMCs from pigs, while none of the CCR3 or CCR9 F(ab)2s stain human PBMCs or Jurkat cells (human T-cell line). These results were not verified in repeat experiments. Repeated transfection of Jurkat cells was performed with results of specific F(ab)2s staining CCR9 transfected Jurkat cells the brightest, CCR10 next and CCR3 poor. Concerns that endogenous expression of human CCRs in Jurkat cells perturbed results necessitated control experiments showing that anti-human CCR3 and anti-human CCR9 mAbs did stain human PBMC, Jurkat cells and pig PBMCs whereas anti-human CCR10 mAbs did not stain any of those cells. Cross-inhibition experiments are planned. Further experiments for anti-CCR3 F(ab)2s and anti-CCR9 F(ab)2s are planned. At the Univ. Bristol a strategy for using the AbD Serotec HUCAL phage display system to identify F(ab)2s reactive with porcine IgE was developed. Sequence analysis of porcine IgE and comparison with six porcine IgGs revealed IgE CH2 was the least conserved domain between each of the six IgGs. As a result, the IgE CH2 domain was used as bait in Fab-bound phage library searches with the result of 10 F(ab)2s as potential reactors with the PoIgE-HuIgG chimeric recombinant proteins. Panels of porcine sera collected weekly following Ascaris suum infection (kindly provided by Dr. J Urban, USDA BARC) will be used to verify native IgE reactivity. A separate test of anti-equine IgE with porcine IgE was proven to be negative (B Wagner, Cornell Univ.) Objective 3: Use reagents produced to develop new assays for swine immune markers. Sensitive sandwich ELISA assays for detecting pg levels of cytokines were established for PoIL-17A (300pg/ml), PoIL-13 (1000pg/ml).and PoCXCL10 (1000pg/ml). The results were not sensitive for PoIL-6 (3000pg/ml) possibly due to the limited number of mAb epitopes developed. Next, for intracellular immune staining, a strategy had to be developed to verify whether cytokine expressing cells could be identified using labeled anti-cytokine mAbs. Certain targets might require pathogen stimulated cells, e.g. PBMC collected from influenza or Ascaris suum infected pigs; others might be expressed by certain cell subsets, e.g., activated macrophages. Thus, marker stimulation conditions were tested and cytokine expression verified if a sandwich ELISA was available. For best sensitivity purified anti-cytokine mAbs were labeled with AF647 and tested on appropriately stimulated cells and costained with T cell anti-CD3, CD4 or CD8 mAbs before analyses using flow cytometry. Results of analyses of anti-porcine IL-17A mAbs were verified on lymphocytes stimulated with PMA and Ionomycin. Several of the tested mAbs showed a clear population (0.6-1.4%) of PE-labeled CD3+ T cells coexpressing IL-17A. Similar experiments were performed with the panel of anti-porcine IFNg mAbs with only 1 showing potential specific results. For anti-porcine IL-13 mAbs results indicate a few reactive mAbs but further studies are required. For anti-porcine chemokine CXCL10 several clones appear to be reactive not with T cells but with the macrophage CD172+ cells. Objective 4: Provide the veterinary community with new commercial reagents and up-to-date information and techniques for their research efforts. Efforts have continued to develop plans for marketing mAb products. As already noted, a commercial source for all expressed immune makers, cytokines and chemokines, is available through Kingfisher Biotech, Inc. Once mAbs became available, tests for potential mAb pairs for multiplex bead quantitative assays were performed with a separate commercial partner. Panels of mAbs were transferred by USDA ARS MTA to the commercial source to test for their development. After testing 5 panels of mAbs they selected pairs of mAb for 4 targets for their new assays. Despite these successful results the USDA ARS technology transfer agreement was never completed due to financial issues. A third company was contacted but was unwilling to provide USDA with a business plan on mAb usage. Alternate sources for commercial transfers of these mAbs are continuing to be explored.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Chase C, Lunney JK. 2019. Swine Immune System. Chapter 16 In: Diseases of Swine (11th Edition) Editors: Zimmerman J, Karriker L, Ramirez A, Schwartz K, Stevenson G, Zhang J. John Wiley & Sons, Inc., Hoboken, NJ. Chap. 16, pp.264-290.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dawson HD, Lunney JK. 2018. Porcine cluster of differentiation (CD) Markers 2018 Update. Res Vet. Sci. 118: 199-246. doi: 10.1016/j.rvsc.2018.02.007.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Dawson HD, Sang Y, Lunney JK. 2020. Porcine cytokines, chemokines and growth factors: 2019 update. Research in Veterinary Science. 131: 266-300. https://doi.org/10.1016/j.rvsc.2020.04.022
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Entrican G, Lunney JK, Wattegedera SR, Mwangi W, Hope J, Hammond JA. 2020. The Veterinary Immunological Toolbox: Past, Present and Future. Frontiers Immunol., section Comparative Immunol. 11: 1651. doi.org/10.3389/fimmu.2020.01651.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Mair KH, Crossman AJ, Wagner B, Noronha L, Stadler M, Gerner W, Saalmueller A, Ladinig A, Lunney JK. 2020. The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) is expressed on the majority of porcine NK cells ex vivo without stimulation. Frontiers Immunol.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Manirarora JN, Walker K, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Lunney JK. 2020. Development and characterization of new monoclonal antibodies against porcine interleukin -17A and interferon-gamma. Frontiers Immunol.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Renukaradhya GJ, Loving C, Kenney S, LaBresh J, Patil V, Byrne K, Walker K, Dai C, Hailstock T, Lunney JK. Development and characterization of new swine immune reagents to understand immune correlates for vaccines, infection, and biomedical research outcomes. The Conference of Research Workers in Animal Diseases (CRWAD) Dec 2020.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lunney JK, Bailey M, Manirarora J, Renukaradhya G,. Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. Development and characterization of immune reagents for swine health, vaccine and disease studies. IVIS 2019, the International Veterinary Immunology Symposium, 13-16 August in Seattle WA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Dawson HD, Lunney JK. Porcine cytokines, chemokines and growth factors: 2019 Update IVIS 2019, the International Veterinary Immunology Symposium, 13-16 August in Seattle WA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Lunney JK, Bailey M, Manirarora J, Walker K, Patil V, Renukaradhya G,. Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. Development and characterization of immune reagents for swine health, vaccine and disease studies. The Conference of Research Workers in Animal Diseases (CRWAD), November 3-5, 2019, Chicago IL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Lunney JK, Bailey M, Manirarora J, Renukaradhya G,. Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. Development and characterization of immune reagents for swine health, vaccine and disease studies. The Conference of Research Workers in Animal Diseases (CRWAD), December 1-3, 2018, Chicago IL
|
Progress 02/15/17 to 02/14/18
Outputs
Target Audience:Our major target audience is swine immunologists. This includes scientists working in swine disease analyses and vaccine development, as well as those involved in developing novel biotherapeutics and nutritional interventions. It includes scientists using the pig as a biomedical model, e.g., for development, obesity, cardiovascular, allo- and xeno-transplantation, and vaccine research. Our efforts are targeted at developing new immune reagents and assays for all of these researchers. Changes/Problems:A formal request has been submitted for a 1 year no cost extension for this grant to continue our efforts and to match up timelines with our UK colleagues. A co-PD, Yongming Sang has successfully moved to Tennessee State Univ. and his remaining subcontract funds transferred from BARC to Tennessee State Univ. Our new coPI, Scott Kenney, Assistant Professor, The Ohio State Univ., has been added. He is working on the pig CD1d project. What opportunities for training and professional development has the project provided?A graduate student and technician have been trained to perform ELISA and flow cytometry assays for screening of pig CD1d specific mouse serum samples and for screening of IFNg mAb at the Ohio State University. A collaborator from the Korean Rural Development Administration, National Institute of Animal Science, Republic of Korea, is training in mAb screening techniques at BARC. A graduate student is performing immunohistochemistry tests of anti-CX3CL1 mAbs at the Univ. of Bristol. How have the results been disseminated to communities of interest? Posters were presented at relevant meetings at the American Association of Immunologists meeting May 12-15 in Washington, DC; at the Conference of Research Workers in Animal Diseases, December 3 -5, 2017, Chicago, IL; at the British Society for Immunology December 4 - 6, 2017 in Brighton, UK.; at the 14th International Xenotransplantation Association (IXA 2017) Congress, September 20 -23, 2017 Baltimore, MD #49; and at the 6th Swine in Biomedical Research Conference (SBR 2017), September 23- 25, 2017, Baltimore, MD #06. Manuscripts for peer review journals are in preparation. What do you plan to do during the next reporting period to accomplish the goals?The contract hybridoma company is preparing for immunizations to produce panels of mAb for several new targets including IgE and IFNb. At BARC further characterization of mAbs will continue, e.g., epitope assignments for anti-CXCL10 and CX3CL1, epitope assignments for new mAbs, and other assays as appropriate. Ohio State scientists will perform more tests of intracellular staining for panels of anti-cytokine mAbs. Once produced, CD1d will be used to produce mAbs. At Tennessee State Univ. functional tests of mAbs against IFNw1 and IFNw5 will be assayed to determine their specificity with the expressed and natural antigens in cell cultures. Scientists at Univ. of Bristol will continue to characterize the anti-CCR mAbs. Gut staining studies will be continued. CCR10 F(ab)2s will be tested as well as titrations for anti-CCR10 F(ab)2s. Dual/tri-staining experiments for anti-CX3CL1 mAbs, anti-CCR3 F(ab)2s and anti-CCR9 F(ab)2s are planned. The coPIs are working with the Veterinary Vaccines network, and UK researchers at Pirbright and Roslin, to develop an updated website to track toolkit/toolbox progress and document availability of reagents and tools for the worldwide veterinary immunology community.
Impacts
What was accomplished under these goals?
For Objective 1, our commercial partner, Kingfisher Biotech, Inc., has cloned and expressed swine proteins using their yeast Pichia pastoris system. Most recently these included swine cytokines [interleukin 28B (IL-28B), IL-29], chemokine (CCL19) and other immunological markers, e.g. annexin, perforin. Efforts to express granzyme are still underway. Once completed the purified proteins are provided to the project for other uses and are available to the community as commercial products. The project team suggests new priorities for protein targets for Kingfisher; it then prioritizes successfully expressed targets for monoclonal antibody (mAb) production. For Objective 2, panels of mAbs have been produced with commercial partners for selected swine cytokines and chemokines in the US and chemokine receptors in the UK. Yeast expressed cytokines, interleukin-6 (IL-6), IL-13, IL-17A and chemokines (CX3CL1, CXCL10), produced by Kingfisher Biotech, Inc., were selected because no pig specific mAbs are yet commercially available. The immunization and fusion for each target resulted in a panel of >10 final hybridoma clones for all but CX3CL1, which had only 3 clones even after 3 fusions. The hybridomas were grown so that mg quantities of the purified mAbs are prepared by the company for further testing. A new set of hybridomas will be initiated in 2018. At BARC ELISAs were used to identify epitope reactivity, based on inihibition assays with biotinylated mAbs and on analyses of mAb cross reactivity on orthologous proteins from other species (supplied by Kingfisher Biotech Inc.). This was completed for IL-6, IL-13, IL-17A and IFNg. At Ohio State Univ. the panel of 9 anti-poIFNg biotin conjugated mAbs was tested for intracellular staining of CD3+ porcine lymphocytes stimulated with PMA and Ionomycin. Of the nine mAb two (1.1 and 35.1) were reactive, Subsequent blocking assays with mAb 1.1 showed that >80% of the specific reactivity to intracellular IFNg was blocked with the respective purified mAb. Cross-inhibition assays between a commercial anti-PoIFNg (P2G10) and 1.1 mAbs revealed absence of any cross-binding indicating that the mAbs each reconized unique epitopes on porcine IFNg. At the University of Bristol, the 3 anti-CX3CL1 mAbs were tested for gut staining. Strong villi staining was observed for one antibody and follicular staining was observed for a different antibody. At Ohio State plans are underway to produce mAb to CD1d (NKT cell target ligand). Computer designed peptides (coupled to KLH) were used as immunogens in CD1d knockout (KO) Balb/c mice. The mouse serum was tested on conventional and CD1d knockout pig peripheral blood mononuclear cells (PBMCs). Some reactivity was seen via ELISA against the synthetic peptides and against conventional pig PBMCs but unfortunately also against CD1d knockout PBMCs. Further boosting of the mice with full length CD1d protein did not improve the immune response. So attempts to clone the CD1d sequence were initiated and were successful with CD1d in frame with enhanced yellow fluorescent protein (EYFP). However, transient expression of transfected Huh7 liver cells and swine testicular cells failed to produce an eYFP signal as evaluated by fluorescence microscopy. Lysates of transfected cells also failed to produce a positive signal in western blotting using an antibody against eYFP. So, at this time there is no expression plasmid that produces full length porcine CD1d. After substantial discussions we have contacted the NIH tetramer facility as they have successfully generated human and mouse CD1d tetramers. They have agreed to synthesize porcine CD1d. We are currently debating whether tetrameric CD1d which is produced by biotinylating CD1d at the tetramer facility, or monomeric CD1d, would be a better option for further immunization studies. If the cloning and expression works the facility will also provide their lentiviral expression vector for new mice immunizations to be performed. At Tennessee State Univ. functional tests have affirmed superiority of porcine IFN-omega (IFNw) subtype in antiviral and inflammatory regulation. Earlier work with a second company had produced a few mAb against IFNw1 and IFNw5. Assays are now underway to determine the mAb specificity with the expressed and natural antigens in cell cultures. More mAbs may be produced in 2018. At the University of Bristol, targets were the chemokine receptors (CCR3, CCR9 and CCR10) and were aimed at producing mAbs against extracellular loop 2 (ECL2) of each chemokine receptor. mAbs have been received for three, two and eight bivalent Fab (F(ab)2) fragments, originating from phage display searches against synthetic, chemically constrained peptides corresponding to ECL2 of CCR3, CCR9 and CCR10, respectively. All CCR3 F(ab)2s and one CCR9 F(ab)2 stain PBMCs from pigs, while none of the CCR3 or CCR9 F(ab)2s stain human PBMCs or jurkat cells (human T-cell line). These results are to be verified in repeat experiments and CCR10 F(ab)2s are yet to be tested. Concerns that endogenous expression of human CCRs may perturb results has necessitated experiments staining with anti-human mAbs. Anti-human CCR3 and anti-human CCR9 mAbs did stain human PBMC, jurkats and pig PBMCs wheras anti-human CCR10 mAbs did not stain jurkats, human PBMCs or pig PBMCs. Cross-inhibition experiments are planned. Limited anti-CCR3 gut staining has been observed, however repeat experiments are required. CCR10 F(ab)2s are yet to be tested. Titrations for anti-CCR10 F(ab)2s and dual/tri-staining experiments for anti-CX3CL1 mAbs, anti-CCR3 F(ab)2s and anti-CCR9 F(ab)2s are planned. A strategy for production of mAbs to IgE was developed. Sequence analysis of porcine IgE and six porcine IgGs revealed IgE CH2 was the least conserved domain between each of the six IgGs. The IgE CH2 domain will be used as bait in Fab-bound phage library searches. In addition, IgE CH2 domain will be expressed by Kingfisher Biotech in yeast and used as the immunogen in mice with our commercial partners. For Objective 3, assays for swine immune markers are being established. These included identifying mAb pairs that work for sandwich ELISAs and multiplex bead-based assays for cytokines and chemokines. The National Pork Board funded the development of the first multiplex assays for cytokines. Tests for multiplex bead quantitative assays are underway with another commercial partner; they have selected pairs of mAb anti- IL-17A, CXCL10 and CX3CL1 for their new assays. Paperwork for transfer of reagents are in progress. Further intracellular staining studies are underway to identify positive mAbs for the 4 cytokines as well as chemokines CXCL10 and CX3CL1 and new targets. For Objective 4 there will be commercial sources for reagents and updates to the community. Kingfisher Biotech, Inc. continues to market expressed cytokines and chemokines. Efforts are underway to develop plans for marketing mAb products. Information has been disseminated to swine researchers via regular updates, Posters were presented at relevant meetings at the American Association of Immunologists meeting May 12-15 in Washington, DC; at the Conference of Research Workers in Animal Diseases, Chicago, IL; at the British Society for Immunology, in Brighton, UK,14th International Xenotransplantation Association (IXA 2017) Congress,Baltimore, MD; and 6th Swine in Biomedical Research Conference (SBR 2017), Baltimore, MD.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Manirarora JN, Bailey M, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L, Lunney JK. 2017. Development of new immune reagents for swine health, vaccine and disease studies. The American Association of Immunologists (AAI) Annual Meeting (IMMUNOLOGY 2017), May 1216, 2017, Washington, D.C. J Immunol May 2017, 198 (1 Supplement) 226.12.
Francis O, Bailey M, Wooldridge L, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Lunney JK. 2017. Development of new immune reagents for swine health, vaccine and disease studies. British Society for Immunology (BSI) Congress 2017, December 4 6, 2017 in Brighton, UK.
Lunney JK, Bailey M, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. 2017. Development and characterization of immune reagents for swine health, vaccine and disease studies. The Conference of Research Workers in Animal Diseases (CRWAD), December 3-5, 2017, Chicago IL.
Lunney JK, Bailey M, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. 2017. Development of new immune reagents for swine health, transplantation, vaccine and disease studies. 14th International Xenotransplantation Association (IXA 2017) Congress, September 20 -23, 2017 Baltimore, MD #49.
Lunney JK, Bailey M, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. 2017. Development of new immune reagents for swine health, transplantation, vaccine and disease studies. 6th Swine in Biomedical Research Conference (SBR 2017), September 23- 25, 2017, Baltimore, MD #06.
|
Progress 02/15/16 to 02/14/17
Outputs
Target Audience:Our major target audience is swine immunologists. This will include scientists working in swine disease analyses and vaccine development, as well as those involved in developing novel biotherapeutics and nutritional interventions. It will include scientists using the pig as a biomedical model, e.g., for development, obesity, cardiovascular, allo- and xeno-transplantation, and vaccine research. Our efforts will be targeted at developing new immune reagents and assays for these researchers. Our website will highlight our plans and progress. Changes/Problems:A co-PD, Yongming Sang moved from Kansas State to TN State. We will request moving his remaining subcontract funds from BARC to TN State in year 3. Scott Kenney, Assistant Professor, The Ohio State University, has been added to the project and is working on pig CD1d project. He will take the place of the late Dr. Kumaran. Thesubcontract with VA Tech was terminated. A one year no-cost extension is requested. This is a US UK collaborative grant. Our UK partners did not start their efforts until close to the beginning of year 2 of the US grant. So to match endpoints [and complete more of our goals] we are requesting this extension. What opportunities for training and professional development has the project provided?A graduate student and technician have been trained to perform ELISA and flow cytometry assays for screening of pig CD1d specific mouse serum samples and for screening of IFNg and β mAb at the Ohio State University. Two technicians (both minorities), a Chinese Visiting graduate student and a USDA Office of Outreach, Diversity, and Equal Opportunity (ODEO) - sponsored summer student worked on this project at USDA ARS BARC in the second year. How have the results been disseminated to communities of interest?Posters were presented at the Conference of Research Workers in Animal Diseases, December 4 - 6, 2016 Chicago, IL, USA; at the British Society for Immunology December 6 - 9, 2016 in Liverpool, UK; and at the International Veterinary Immunology Symposium 2016 (IVIS 2016), 16-19 August 2016, in the Gold Coast, Australia. A poster will be presented at the American Association of Immunologists meeting May 12-15, 2017 in Washington, DC. What do you plan to do during the next reporting period to accomplish the goals?For year 3, , Kingfisher Biotech is planning to express granzyme, perforin, IL-27, IL-33, IL-35, and CCL27. Univ. of Bristol is planning efforts for immunoglobulin E (IgE). A subset of these molecules will be used to immunize mice to produce mAbs specific for several epitopes of each product. These mAbs will then be used to develop quantitative assays for these targets. The first contract hybridoma company is purifying panels of mAb products against 2 expressed chemokines, CX3CL1 and CXCL10. Growth of mAb panels against IL-17A and CXCL10 are in process. Plans with the second contract hybridoma company have been terminated due to poor productivity. At Ohio State tests of mouse anti-peptide bleeds are underway for affirming specific reactivity with internal or external staining of different cell populations derived from CD1d KO and convention pig derived PBMCs and thymocytes. We expect to increase the number of mAbs reactive with cell surface, or CD, antigens. These are expected to include (via our UK colleagues):integrins [CD49D (integrin a4), integrin b7 and the a4b7 heterodimer], CD205, chemokine receptors [CD193/CCR3, CCR6, CDw199/CCR9, CCR10, CD186/CXCR6, CX3CR1] and other receptors, e.g., EMR1. Our PI at Kansas State Univ. has just moved to Tennessee State Univ. and is establishing his research lab there. Members of his lab remain at Kansas State and will test mAbs anti-IFNs using mammalian systems for specific reactivity with the mammalian expressed targets. The coPIs will develop a single website to track our progress for developing reagents and provide updates to the community.
Impacts
What was accomplished under these goals?
For Objective 1, our commercial partner, Kingfisher Biotech, Inc., has cloned selected swine cytokines [interleukin 28B (IL-28B), IL-29], chemokines (CCL19) and other immunological markers, e.g. annexin, perforin. They express the proteins using their yeast Pichia pastoris system. The purified proteins are provided to the project for other uses and to the community as commercial products. The project team suggests new priorities for protein targets for Kingfisher; it then prioritizes successfully expressed targets for mAb production. The team may affirm bioactivity of these products. For Objective 2, panels of monoclonal antibodies (mAbs) are being produced for selected cell surface or CD markers and swine cytokines, chemokines and receptors. The team has primarily used expressed cytokines (e.g., interleukin-6 (IL-6), IL-13, IL-17A) and chemokines (CX3CL1, CXCL10) from Kingfisher Biotech, Inc. for which no pig specific mAbs are yet commercially available. Using one commercial partner each immunization and fusion is expected to result in a panel of hybridoma clones for each target. All but CX3CL1 resulted in panels of >10 clones; CX3CL1 had only 3 clones even after 3 fusions. The hybridomas are cloned, grown in quantities so that mg quantities of the purified mAbs are supplied by the company for further testing. At BARC ELISAs are used to identify epitope reactivity by inhibition assays as well as analyses of mAb cross reactivity on other species proteins. This has been completed for IL-6, IL-13 and IFNg. A panel of anti-IL-17A mAbs is almost ready. It should be noted that a 2nd contractor was used to produce mAbs against IL-17A, IL-17F and several IFNs. Rather than a panel of hybridomas only a few clones were produced and some of those were lost during recloning. It was therefore decided to stop providing that company with new proteins and to use the successful company for future fusions. At Ohio State plans are underway to produce mAb to CD1d (NKT cell target ligand). They used computer designed peptides (coupling to KLH) as immunogens in CD1d knockout (KO) Balb/c mice. Sera from these mice have been screened for reactivity on the uncoupled (no KLH)peptides and are in process of being screened on cells from CD1d KO pigs provided by Dr. J Driver at U FL. At the University of Bristol, targets are the chemokine receptors (CCR3, CCR9 and CCR10). mAbs will be targeted against extracellular loop 2 (ECL2) of each chemokine receptor. mAbs will be derived from Fabs retrieved from an antibody phage-display library using peptide mimics corresponding to each ECL2. Scrambled (rearranged, but identical length) peptides will be used to block non-specific selection. Peptide mimics have been sent to a commercial partner and antibody phage library searches are underway. Using a lentiviral expression system, full length CCR3, CCR9 and CCR10 receptors have been expressed in the jurkat T-cell line for screening of positive hits. Expression of these receptors is currently being optimized. Wild type jurkat T-cells will be used for negative screening. Additionally, a strategy for production of mAbs to IgE will use the second IgE constant heavy domain (CH2). Sequence alignments reveal strong difference between IgE CH2 and all IgG immunoglobulin constant heavy domains, therefore mAbs will be targeted against IgE CH2. Expression of monomeric IgE CH2 will be attempted using a bacterial expression system. As with the above chemokine receptors, mAbs will be derived from Fabs retrieved from an antibody phage library using recombinant IgE CH2. For Objective 3, assays for swine immune markers are being established. These will include identifying mAb pairs that work for sandwich ELISAs and multiplex bead-based assays for cytokines and chemokines. The National Pork Board funded the development of the first multiplex assays for cytokines. An MTA with another commercial partner is planned to develop a broader panel of multiplex assays. Other targets will be mAb reactive for intracellular staining for these expressed immune proteins or for cell receptor binding/blocking. For Objective 4 there will be commercial sources for reagents and updates to the community. Kingfisher Biotech, Inc. will continue to market expressed cytokines and chemokines. Information has been disseminated to swine researchers via regular updates, Posters were presented at relevant meetings at the British Society for Immunology December 6 - 9, 2016 in Liverpool, UK; at the Conference of Research Workers in Animal Diseases, December 4 - 6, 2016, Chicago, IL, USA; and at the International Veterinary Immunology Symposium 2016 (IVIS 2016), 16-19 AUGUST 2016, in the Gold Coast, Australia. A poster will be presented at the American Association of Immunologists meeting May 12-15 in Washington, DC.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lunney JK, Bailey M, Crossman A, Manirarora JN, Renukaradhya G, LaBresh J, Sang Y, Wooldridge L. 2016. US-UK Collaborative Swine Immune Toolkit Initiative: Development of new immune reagents for swine health, vaccine and disease studies. International Veterinary Immunology Symposium 2016 (IVIS 2016), 16-19 August 2016, Gold Coast, Australia
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Francis O, Bailey M, Wooldridge L, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Lunney JK. 2016. Development of new immune reagents for swine health, vaccine and disease studies. British Society for Immunology (BSI) Congress 2016, December 6-9, Liverpool, UK.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Lunney JK, Bailey M, Manirarora JN, Renukaradhya G, Kenney S, LaBresh J, Sang Y, Francis O, Wooldridge L. 2016. Development of new immune reagents for swine health, vaccine and disease studies. The Conference of Research Workers in Animal Diseases (CRWAD), December 4-6, Chicago IL.
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Progress 02/15/15 to 02/14/16
Outputs
Target Audience:Our major target audience is swine immunologists. This will include scientists working in swine disease analyses and vaccine development, as well as those involved in developing novel biotherapeutics and nutritional interventions. It will include scientists using the pig as a biomedical model, e.g., for development, obesity, cardiovascular, transplantation, and vaccine research. Our efforts will be targeted at developing new immune reagents and assays for these researchers. Our website will highlight our plans and progress. Changes/Problems:A co-PD, Dr. Elankumaran Subbiah, Virginia-Maryland Regional College of Veterinary Medicine, died in Sept. 2015. We requested that the objective work he was doing and the corresponding unspent funding ($31,113.41) be sent back to USDA ARS BARC. A new co-PI at Ohio State will be recruited to take over Dr. Subbiah's work in 2016. A 2nd co-PD, Yongming Sang is moving from Kansas State to TN State. We will request moving his subcontract from Kansas State University to TN State in 2016. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Posters were presented at the European Veterinary Immunology Workshop (EVIW) Meeting. 2-4th September 2015, Vienna Austria and at the Conference of Research Workersin Animal Diseases (CRWAD), 6 Dec., 2015, and Associated NIFA PD meeting on Dec.4, 2015, in Chicago IL USA. What do you plan to do during the next reporting period to accomplish the goals?The contract hybridoma producer has produced panels of monoclonal antibody (mAb) products against the expressed cytokines IL-6, IL-13, and interferon-gamma (IFNg). Specificity screening was initiated at the company; the coPIs will test the mAbs for intracellular staining and for use in generating new assays for marker quantitation. At Ohio State members of the team will test the mAbs for specific reactivity with internal or external staining of different cell populations to affirm reactivity. A new coPI, Scott Kinney, will join the team in July 2016. He will design efforts to produce anti-porcine CD1d mAbs. At Kansas State members of the team will clone and express interferon targets using mammalian systems, submit them for immunizations and then test the resultant mAbs for specific reactivity with the mammalian expressed targets. The coPIs will develop a single website to track our progress for developing reagents and provide updates to the community.
Impacts
What was accomplished under these goals?
For Objective 1 our commercial partner, Kingfisher Biotech, Inc., has cloned selected swine cytokines, chemokines and other immunological markers, e.g. annexins. They express the proteins using their yeast Pichia pastoris system. The purified proteins are provided to the project for other uses and to the community as commercial products. The project team may affirm bioactivity of these products and integrate prioritized ones into mAb production. For Objective 2 panels of monoclonal antibodies (mAbs) will be produced under contract for selected cell surface or CD markers and swine cytokines, chemokines and receptors. This process has started as there are already expressed cytokines (e.g., interleukin-6 (IL-6), IL-17A) at Kingfisher Biotech, Inc. for which no panel of mAb is yet available. Each immunization and fusion is expected to result in a panel of hybridoma clones for each target. These will enable the team to identify epitope reactivity and utility for different assays (verified in Objective 3) and use advanced UK immunofluorescence screening techniques to identify molecular interactions in tissues. For Objective 3 new assays for swine immune markers are being established. These will include identifying mAb pairs that work for ELISA and multiplex bead-based assays for cytokines and chemokines. The National Pork Board funded the development of the first multiplex assays for cytokines. Other targets will be mAb reactive for intercellular staining for these expressed immune proteins or for cell receptor binding/blocking. For Objective 4 there will be commercial sources for reagents and updates to the community. Kingfisher Biotech, Inc. will continue to market expressed cytokines and chemokines; each hybridoma producer will serve as a source for mAb products. Information will be disseminated to swine researchers via regular updates, e.g., posters at relevant meetings as well as publications.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Lunney JK, Bailey M, Crossman A, Renukaradhya G, Labresh J, Elankumaran S, Sang Y, Wooldridge L. 2015. US-UK Collaborative Swine Immune Toolkit Initiative: Development of new immune reagents for swine health, vaccine and disease studies. European Veterinary Immunology Workshop (EVIW) Meeting. 2-4th September 2015, Vienna Austria
Lunney JK, Bailey M, Crossman A, Renukaradhya G, Labresh J, Elankumaran S, Sang Y, Wooldridge L. 2015. US-UK Collaborative Swine Immune Toolkit Initiative: Development of new immune reagents for swine health, vaccine and disease studies. CRWAD, 6 Dec. 2015, Chicago IL USA.
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