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US Veterinary Immune Reagent Network Cynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Project.

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Presentation on theme: "US Veterinary Immune Reagent Network Cynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Project."— Presentation transcript:

1 US Veterinary Immune Reagent Network Cynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Project Directors Cynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Erin Bromage, University of Massachusetts Dartmouth John 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 Funded by: USDA-CREES ; USDA-NIFA

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: mAb pairs for ELISA, multiplex and ELISpot assays (in some cases this may include polyclonal Abs); single mAb for intracellular staining for cytokines mAbs to Igs isotypes and IgG subclasses 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 Proteins expressed in yeast Note: not all targets in list were requested for all species Those check-marked in Red have ELISAs developed by KingfisherBiotech called VetSets Cytokine/ chemokineCattleSwineHorsePoultry IL-1β & α √√turkey IL-2 √√√ IL-4 √√√√ IL-5 √√ IL-6 √√√√ IL-8 √√√turkey IL-10 √√√ IL-13 √√√In progress IL-15 √√√ IL-16 √ IL-17A √√√ IL-17F In progress√√ IL-18 √√ IL-21 √√ IL-22 √√ IFNα √√√ IFNβ √√√ IFNγ √√√√ CCL2 √√√ CCL3 √√ CCL4 √√√ CCL5 √√√ CCL20 √ CCL11 & CXCL9 √√ CXCL10 √√√ CXCL11 √√ TNFα √√√√ (TNF-SF) GM-CSF √√ Progress US-VIRN target

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 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 VIRN:Catfish Species Report

12 Catfish Progress & Planning

13 Anti-IgD (IgG 1  )  and Anti-IgL  (IgM  ) mAb Available mAbs Anti-IgM Anti-IgD Anti-IgL  FAnti-IgL  Anti-IgL  G B cell subpopulations IgM + /IgD + IgM + IgD +

14 Anti-TCR alpha (IgG 1  ) mAb T cell line TS32.15 PBL CRP % 20 % PBL CRP 16 Anti-TCR alpha

15 Trout Presented by Erin Bromage

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

17 Anti –IgD mAbs

18 Anti-CD3e mAbs

19 Anti-IL1B-2 mAb

20 Chicken

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

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

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 23

24 IL-1β Western IL IL-6 IFN-γ mAb #1 #2 IL-18 ( kDa) Flow cytometry CD25 CD83 C A B Flow cytometric analysis of mAbs to chCD25 (A), CD80 (B) and CD83 (C) Using CHO-chicken cells

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

27 Horse

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

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

30 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 Characterization ongoing: a nti-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) Monoclonal antibodies to equine cytokines, chemokines and cell surface molecules developed at Cornell

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

32 Anti-equine CD25 mAb PBMC, 48h, medium CD4 CD25 PBMC, 48h, PWM CD4 CD25 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 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 mAbs produced Anti-IL-17A mAbs have been also tested now and work well for ELISA, Multiplex assay and FACS.

35 Swine

36

37

38 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 CCL2 to multiplex * Lawson S, Lunney JK, et al. Vaccine 32: , **Wagner, Lunney et al. in preparation

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

40 Cattle

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

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

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

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

45 Mouse titers to Yeast (KF)-expressed bovine CCL2

46 WHERE CAN I GET THESE REAGENTS? GO TO FOR ADDRESS OF SPECIES COORDINATORSwww.vetimm.org KINGFISHER From Cornell University Animal HealthDiagnostic Cntr

47 The Animal Health Diagnostic Center is the NY State Veterinary Diagnostic Laboratory Equine 5-plex cytokine assay


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