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US Veterinary Immune Reagent Network

Project Directors Cynthia Baldwin, University of Massachusetts Amherst Eva Bengten , University of Mississippi Medical Center

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US Veterinary Immune Reagent Network

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  1. Project DirectorsCynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Erin Bromage, University of Massachusetts DartmouthJohn Hansen, WFRC-USGS-Biological Resources Division Joanna LaBresh, Kingfisher Biotech Hyun Lillehoj, USDA-ARS Beltsville Joan Lunney, USDA-ARS Beltsville Bettina Wagner, Cornell University Melanie Wilson, University of Mississippi Medical Center www.vetimm.org US Veterinary Immune Reagent Network Funded by: USDA-CREES 2006-2009; USDA-NIFA 2010-2014

  2. Objective A major obstacle to advances in veterinary immunology and disease control is the lack of sufficient immunological reagents specific for ruminants, swine, poultry, equine and aquaculture species. The goals of the project are to develop sets of reagents to begin to address this dearth.

  3. Targets of US-VIRN • Cytokines and chemokines: bioactive recombinant proteins as well as Abs to them • Ab to cytokines & chemokines: mAbpairs for ELISA, multiplex and ELISpot assays (in some cases this may include polyclonal Abs); single mAb for intracellular staining for cytokines • mAbs to Igsisotypes and IgGsubclasses • mAb anti-CD molecules and T cell receptors: mAb reactive with native molecules and used to stain viable cells for flow cytometric analyses including cell sorting to purify cells by flow cytometry or magnetic beads; for anti-cytokine receptors mAb that block function and signaling

  4. Kingfisher Biotech Presented by Joanna LaBresh

  5. Kingfisher Biotech • 14 Species • Bovine, Canine, Catfish, Chicken, Dolphin, Equine, Feline, Guinea Pig, Mouse, Ovine, Rabbit, Rat, Swine, Turkey • Product Types • Proteins (>175), Antibodies(>140), ELISA Pairs, (>50) ELISA Sets (25)

  6. Proteins • Expressed in yeast because of the advantages of yeast • Expressed into media • Properly folded • Post-translationally modified • No endotoxin • We never use tags

  7. Progress US-VIRN target Proteins expressed in yeastNote: not all targets in list were requested for all species Those check-marked in Red have ELISAs developed by KingfisherBiotech called VetSets

  8. Example: ELISA kit results

  9. New Products • Our pipeline is dynamic • We receive a couple of requests a week. • We are happy to hear what products our customers would like to further their research

  10. Catfish Presented by Mel Wilson

  11. VIRN: Catfish Species Report For catfish, the highest priority reagents have been and are mAbs that recognize B and T cell subsets and macrophages. Antibodies to inflammatory cytokines have also been requested. Our goal is to prove specificity by FACS, western blotting, and immunoprecipitation with peptide sequencing. Peer review publications: 8 Abstracts: 16 Oral presentations:17

  12. Catfish Progress & Planning

  13. Anti-IgD (IgG1k)and Anti-IgL s (IgMk) mAb B cell subpopulations IgM+/IgD+ IgM+ IgD+ Available mAbs Anti-IgM Anti-IgD Anti-IgL k F Anti-IgL s Anti-IgL k G

  14. Anti-TCR alpha (IgG1k) mAb T cell line TS32.15 PBL CRP 16 PBL CRP 14 20 % 19 % Anti-TCR alpha Anti-TCR alpha Anti-TCR alpha

  15. Trout Presented by Erin Bromage

  16. Trout Progress & Planning Full length cDNA Protein produced Mice Immunized Polyclonal sera reactivity mAb reactivity Assay development Molecule mAb Fusion IgL1 IgL2 IgL3 LCK CD3e CD8 IgD secIgM IgT CD79α CD83 CD80 CD4 IL1B-2 CD4rel TCRβ CXCR4 IL6 C3d TCRβ IgL4 Current Position (April 2011) Years 3-4 Seeking alternative external funding or collaborations

  17. Anti –IgD mAbs

  18. Anti-CD3e mAbs

  19. Anti-IL1B-2 mAb

  20. Chicken

  21. Chicken Progress & Planning Assay development Full length cDNA Protein produced Mice Immunized Polyclonal sera reactivity mAb Fusion mAb reactivity Molecule IL-1β IL-2 IL-4 IL-6 IL-7 IL-8 IL-10 IL-12p35 IL-12p40 IL-13 IL-15 IL-16 IL-17A IL-17F IL-18 IL-21 IL-22 CD25 CD80 CD83 CD86 Current Position At the end of Mar 2013 At the end of April 2014

  22. Chicken Progress & Planning Assay development Full length cDNA Protein produced Mice Immunized Polyclonal sera reactivity mAb Fusion mAb reactivity Molecule CCL4 CCL20 CXCL14 GMCSF IFN-α IFN-γ LITAF LT MIF NK lysin TGFB4 TL1A IL1R1 IL-5R IL-7R IL-10R-β IL17R IL21R Β-defensin8 VEGF Seeking external funding Current Position At the end of Mar 2013 At the end of April 2014

  23. Reagents Completed through to mAbs • 11 mAbs to chicken protein - IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γ • Test method Western: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γ Flow cytometry: CD25, CD80, CD83, CD86 Tissue staining: CD25, CD80, CD83 Neutralizing functional assay: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, IFN-γ

  24. Flow cytometric analysis of mAbs to chCD25 (A), CD80 (B) and CD83 (C) Using CHO-chicken cells • Western • Flow cytometry IL-1β IL-6 IL-8 B A 148 98 64 50 36 22 148 98 64 50 36 22 148 98 64 50 C mAb #1 #2 IFN-γ CD25 CD83 IL-18 ( kDa) 148 98 64 50 36 16

  25. Tissue staining with mAbs to CD25, CD80 & CD83 Immunolocalization of chCD25 in tissues. Bursa of Fabricius (A), spleen (B), and intestinal duodenum (C) Expression of chCD80 on LPS-stimulated dendritic cells. Immunolocalization of chCD83 in lymphoid organs. (A) Caecal tonsil, (B) bursa of Fabricius, and (C and D) spleen

  26. Functional assays with mAbs (anti-IL-18, CD25, CD80 & IL-1) Effect of anti-chCD80 mAb on IL-2 driven lymphoblast cell proliferation. Neutralization of IL-18 bioactivity by chIL18 mAbs. Inhibition by mAb to CD25 on IL-2-dependent splenocytes proliferation. Functional activity of chIL-1β on thymus lymphocyte proliferation and its mAb’s neutralization effects.

  27. Horse

  28. Horse Progress &Planning: Cytokines & Chemokines COMPLETED: IL-2 IL-4 (mamm) IL-5 IL-6 IL-10 (mamm) IL-17A IFN- (mamm) CCL2 CCL3 CCL5 CCL11 Expression vector/ Transfectant Full length cDNA Protein produced Mice immunized mAb reactivity Assay development Molecule mAb Fusion IL-4 IL-2 CCL2 CCL3 CCL5 IL-6 IL-5 IL-17A CCL11 Current Position (1/2012) GM-CSF Continued during this funding period IL-1 IL-8 IL-13 No mAbs with E. coli or yeast expressed proteins IL-15 CXCL9 CXCL10 Mabs to yeast protein did not react with native cytokine IL-12 IL-18 Yeast protein development/ mAb development not planned (antibodies exist) TGF- IFN- IFN- Used both yeast and mammalian-expressed cytokines & chemokines for immunization

  29. Horse Progress & Planning: Cell Surface Molecules Protein produced Full length cDNA Protein purified Mice Immunized mAb Fusion mAb reactivity flow cytometry Molecule CD23 CD25 CD28 Completed:CD14 CD23 CD25 CD16 CD40 FcRI TCR CD40 FcRI TCR TCR TCR IgD CD19 CD16 Foxp3 CD34 CD105 NCR2 NCR3 IFNAR TLR2 Continued Current Position (1/2012) Procedure repeated Done by collaborator lab

  30. Monoclonal antibodies to equine cytokines, chemokines and cell surface molecules developed at Cornell Characterized and published antibodies (available through Cornell): Anti-equine IL-4 Anti-equine IL-10 Anti-equine IFN- Anti-equine CD14 Anti-equine CD23 Characterized antibodies (available soon): Anti-equine IL-2 Anti-equine CCL2 Anti-equine CCL3 Anti-equine CCL5 Anti-equine L-17A Anti-equine CD25 Anti-equine CD16 http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm http://www.vetimm.org Characterization ongoing: anti-IL-5, anti-CCL11 Equine cytokine multiplex assay (IL-4, IL-10, IL-17, IFN-, IFN-) available through the Animal Health Diagnostic Center at Cornell (ahdc.vet.cornell.edu)

  31. Anti-equine CD14 mAbs Magnetic cell sorting, CD14 mAb 105 CD14 mAb 59 (lane 2) Mammalian expressed equine CD14 was used for immunization (Kabithe et al. 2010, Vet Immunol Immunopathol)

  32. Anti-equine CD25 mAb 3 mAb clones, tested on PBMC of various horses in 2011, given to two equine labs outside US-VIRN for additional specificity testing who confirmed our results PBMC, 48h, medium PBMC, 48h, PWM CD25 CD25 CD4 CD4 Mammalian expressed equine CD25 was used for immunizations

  33. Anti-equine IL-17A mAbs Equine PBMC, non-stimulated or stimulated with PMA/ionomycin, stained and analyzed by flow cytometry Anti-equine L-17A mAbs were made using yeast expressed IL-17A

  34. Applications for anti-equine cytokine mAbsproduced Anti-IL-17A mAbs have been also tested now and work well for ELISA, Multiplex assay and FACS.

  35. Swine

  36. Expanded Swine cytokine Fluorescent Microsphere Immunoassay (FMIA) (magnetic bead assays) Lab developed FMIA* 6-plex detected IL-1b, IL-4, IL-8, IL-10, IL-12, IFNa; As a result of US Toolkit efforts** have recently added CCL2to multiplex * Lawson S, Lunney JK, et al. Vaccine 32: 5383-5391, 2010. **Wagner, Lunney et al. in preparation

  37. Increased levels of serum CCL2 were associated with pigs which had higher serum PRRS virus burden P<0.05 repeated measures combined with 0-21 dpi viral load [low vs high] P Souza, Araujo, Lunney et al. in preparation

  38. Cattle

  39. Cattle Progress &Planning: Cytokines & Chemokines Expression vector/ Transfectant Bioactivity determined Full length cDNA Protein produced Mice immunized mAb reactivity Assay development Molecule mAb Fusion CCL2 CCL5 CCL11 CXCL9 CXCL10 CXCL11 IFN-α IFN- IFN-β IL-1β IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-12p35 IL-12p40 IL-13 IL-15 IL-17A IL-17F IL-18 TNFα Current Position Continuing Commercially available With new funding

  40. Cattle Progress & Planning: Cell Surface Molecules Full length cDNA Protein produced Protein Purified Mice Immunized mAb Fusion mAb reactivity Molecule TCRγ-C3 TCRδ–V1 TCRβ TCR α IL-23R IL-10R CCR7 TCRδ-V2 TCRδ-V3 TCRδ-V4 TCRγ-C5 CD107a CD207 Continuing in current grant Current Position With new funding

  41. Monoclonal antibodies to bovine IL-17F using mammalian-expressed protein – 3 parent clones available

  42. Monoclonal antibodies made to bovine IL-17A using mammalian-expressed protein: Subclones of 6F2

  43. Mouse titers to Yeast (KF)-expressed bovine CCL2

  44. WHERE CAN I GET THESE REAGENTS? • GO TO www.vetimm.org FOR EMAIL ADDRESS OF SPECIES COORDINATORS • KINGFISHER BIOTECH @ www.kingfisherbiotech.com • From Cornell University Animal HealthDiagnostic Cntr http://ahdc.vet.cornell.edu/sects/Serol/

  45. http://ahdc.vet.cornell.edu/sects/Serol/ Equine 5-plex cytokine assay The Animal Health Diagnostic Center is the NY State Veterinary Diagnostic Laboratory

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