Swine Toolkit Progress for the US Veterinary Immune Reagent Network

Agricultural Research 1 1 1 2 2 2 3 4 Service JK Lunney , P Boyd , A Crossman , K Araujo, J LaBresh , L. Kakach , Y Sullivan , B Wagner , H Dawson , C Chitko-McKown 5, D Tompkins 6, E Hudgens 6, C Baldwin 6 1APDL, BARC, USDA, Beltsville, MD; 2 Kingfisher Biotech, St. Paul, MN; 3 Cornell University, Ithaca NY; 4 DGIL, BARC, USDA, Beltsville, MD; 5 USDA ARS MARC, Clay Center NE; 6 University of Massachusetts, Amherst MA

Abstract BACKGROUND The US Veterinary Immune Reagent Network (US VIRN, www.vetimm.org) was established US VIRN: Swine Toolkit Planning - Update 12/11 Chemokines, Information on VIRN progress is summarized by species at www.vetimm.org on the various web pages and in attached PDFs on the web page as FASTA files, alignments of being expressed, commercially available to address the lack of immunological reagents specific for veterinary species. Efforts are Full length Protein Protein Mice mAb mAb Assay Molecule reagents for swine, protocols for bioassays of cytokines and chemokines, primary references for the cloning targeted at swine, ruminants, poultry, equine and aquaculture species. Our goal is to cDNA produced Purified Bioactivity Immunized Fusion reactivity development and detection for each species. produce reagents that function in ELISA, Luminex assays, ELISPOT and flow cytometric CCL2 Chemokines & Cytokines Following the completion of the gene cloning phase for the cytokines and chemokines, a CCL3L1 large scale alignment and annotation of the expressed sequences was done to confirm the correctness of the applications. In the last year recombinant chemokines (CCL3L1, CCL4, CCL5 and CCL20) and CCL4 sequences before expression at Kingfisher (KF). These are shown on the website. Whole sequence included cytokines [interleukin-6 (IL-6) and IL-22] were expressed in Pichia, purified and all but IL- CCL5 CCL20 signal/leader sequence while those designated as KF are ready for expression in the yeast system in most cases 22 shown to be bioactive using chemotaxis, upregulation of marker expression or cell CCL25 without signal sequence or pro-peptides. stimulation assays. We have also cloned, expressed and proven bioactivity of swine CCL28 All these genes are available for distribution to the scientific community upon request and we are investigating immunoregulatory cytokines, IL-17A and IL-17F. Hybridoma fusions for monoclonal CXCL9 feasibility of deposition in gene banks. CXCL10  Virtually all sequences have been deposited in GenBank. CXCL11 antibodies (mAb) to CCL3L1, IL-6, IL-13, IL-17A, interferon-alpha (IFNα) and IFNβ are  Kingfisher (KF) biotech is in the process of expressing all these in Pichia and those expressed have been sent to underway at Univ. Massachusetts. A sensitive fluorescent microsphere, Luminex bead, IFNa1,6 the various species coordinators for biotesting. immunoassay for CCL2 was developed with US VIRN produced mAb and included in the 8- IFNb  The bioactive are commercially available (see Kingfisher website www.KingfisherBiotech.com) and a g plex swine cytokine assay. At Cornell Univ. a fusion protein expression system was used to IFN limited amount directly distributed to scientists. TNFa  A selected number will be used for producing mAbs by US-VIRN. generate material for immunizations for swine T cell receptors, TCRαβ; hybridoma fusions IL-6 are continuing. Additional fusions will target IFNαR, CD19, and NK cell markers, NKp36 Cell Surface Molecules These genes were largely cloned by US-VIRN and are being transfected into CHO cells at IL-7 Cornell by B Wagner while the fish labs have produced their cell surface proteins in bacteria or mammalian cells. IL-9 (NCR3) and NKp44 (NCR2). The US VIRN website www.vetimm.org has a progress update All will be used for production of mAb. IL-13 for swine as are all bioassay methods and gene sequences. Since many swine cytokine and Gene sequences have been/will be deposited into GenBank. IL-15 CD reagents are available commercially the website includes a listing of those reagents and IL-17A Most genes are available upon request. their sources. Products developed in this proposal are available to collaborators and have IL-17F All the proteins in this category have been, are in the process of or will be used for producing mAbs. IL-21 Transfected cell lines will also be available for testing other potential cross-reactive mAbs. been made commercially available through Kingfisher Biotech, Inc. IL-22 http://www.kingfisherbiotech.com/. This project was funded by USDA NIFA proposal IL-23A Monoclonal antibodies (mAbs) : mAb will be made at UMass against cytokines or chemokines that have shown IgG1 bioactivity in the Species labs. In addition, the cell surface molecules, e.g., T cell (TCR) proteins being #2006-35204-16880, renewal #2010-65121-20649, US DHS IAA #HSHQDC10X00021 IgG2 expressed at Cornell in the Wagner Lab will be used for mAb at Cornell. and USDA ARS project 1265-32000-098 funds. IgG3 These are being continually re-prioritized by the species coordinators in conjunction with the scientists in field; IgG5 the current version is shown. Current Position (Nov. 2011] VIRN Renewal 2011-12 Plans 2011-12 DHS/NIFA funding plans Future Year Plans Not planned The hybridoma cell lines that produce the mAbs will be deposited in cell banks for distribution upon request and U.S. Veterinary Immune Reagent Network Team distributed to a limited number of scientists directly by Network members; the mAb they produce will be (US VIRN) available through commercial companies including Kingfisher.

Overall Background: This is a multi-species immune reagent grant from USDA NIFA AFRI for development of a US Veterinary Immunological Reagents Network, which will support US VIRN: Swine Toolkit Planning - Update 12/11 Cell Surface Proteins Cloning of porcine immune genes: Regulatory cytokine IL-17F; Full length Protein Peptide Mice mAb mAb mAb Epitope immunological reagents specific for ruminants, swine, poultry, equine and aquaculture species Molecule cDNA expressed Identified Immunized Fusion reactivity defined Natural Killer (NK) cell markers NCR2 (NKp44), NCR3 (NKp30)

to advance veterinary immunology and disease control. (www.vetimm.org) TCRa Bioinformatics: Search available databases for swine ESTs or genomic sequences; use comparative Swine Plans: The emphases for swine will be on developing and characterizing bioactive TCRb (human/bovine/mouse) approaches to identify unannotated genes. Searches revealed that no cDNA or immune proteins, cloned cytokine and chemokine proteins, as well as monoclonal antibodies ESTs existed for IL-17F or NCR2. NCBI Gene had predicted open reading frame sequences based on (mAbs) to these proteins and their receptors. [See 11/10 updates on poster.] Examples of IL-4Ra other species. There was a cDNA for NCR3. IL-13Ra our current approaches for each type of reagent is given on this poster as well as the IL-17Ra Cloning gene for expression: Primer Premier software was used to design 2 sets of primers for each current priority list. IFN Ra gene for best results and potential nested PCR for lowly expressed RNAs. cDNA used for amplification CXCR3 Expected Applications: These reagents will be used to: (1) evaluate changes during disease CXCR5 was a pooled cDNA from Toxoplasma gondii infected pigs. Various immune tissues were used. The IL- and following vaccination and (2) give scientists the ability to manipulate these cell CD45RO 17F was cloned from cDNA derived from PBMC stimulated with ConA, IL-6 and TGF- 1 for 5 days. populations to evaluate their roles in protective immunity as well as in immunopathology. CD1c The IL-17F product was a clean band on gels. The NCR2/3 PCRs was performed at 55 or 60oC CD19 annealing. The NCR genes had high GC content so DMSO was added. To get the final clones various NKp30 combinations of primers and gel extraction of early PCRs was required to get a clean product for NKp44 NKp46 Austria sequencing and plasmid packaging. PCR fragments were gel purified, inserted into TOPO2.1 cloning vector, PCR verified for the correct size insert and then sequenced. The sequence closest to the Swine Cytokine FMIA predicted sequence was chosen. Po NCR2 has 2 changes that translate into 2 amino acids changes, indicating a SNP. (4 sequenced clones affirmed this sequence change). Luminex or fluorescent microbead immunoassay (FMIA) Current Position (Nov. 2011] VIRN Renewal 2011-12 Plans 2011-12 DHS funding plans Renewal Future Year Plans Not planned The sequences were submitted to NCBI, trimmed of primers. rPoIL17F (HM189210); • Multiple targets (many answers per sample) Po NCR3 (HQ658981) Good Homology to EU282355 Cytokine production by C2 macrophages stimulated with rPoIL-17Po NCR2 (submitted to NCBI) Predicted from genomic DNA: XM_0019246 • Broad dynamic range (3-4 log pg/ml) sensitivity • Flexibility for targets (assay 1-2 or multiplex) Expression: IL-17F has been inserted into a yeast expression vector at Kingfisher and expressed. It still requires • Small sample volume required (50ul) 10000 Bioactivity of rPoIL-17A purification before it can be sent to Univ. MA for immunization and mAb production. • High throughput screening for multiple samples The plasmids with the NK markers NCR2 & NCR3 are being sent to Cornell to be put into a mammalian 8000 rPoIL-17F rPoIL-17A and expression system for immunization and mAb production targeting the extracellular portion of the Swine Cytokine Multiplex FMIA rPoIL-17F 6000 proteins. Screening will be done by flow cytometry on fresh PBMCs looking for NK cells. 8-plex initial set of cytokine targets The porcine macrophage cell 4000 Innate: IL-1β, IL-8, IFN-α, TNF-α line CΔ2 (Chitko-McKown, [pg/ml] IL-6 2000 Screening New Hybridomas Regulatory: IL-10 ARS MARC) was stimulated for anti-IL-17A mAb Reactivity Th1: IL-12, IFN-γ for 24 hour with yeast rPoIL- 0 0.001 0.01 0.1 1 10 100 1000 • Purified IL-17A proteins used for mouse immunizations at Th2: IL-4 Problem commercial mAb reagent availability 17A or rPoIL-17F (Kingfisher IL-17 [ng/ml] #RP0128S-025 and RP0291S-025) UMass Amherst; combine proteins (IL-17A + CCL3L1) for VIRN assay: CCL2 chemokine; Additional targets in development: IL-17A, IFN-b Results show that rPoIL-17A more effective immune stimulation. and, to a lesser degree, • Affirmed mouse sera for positive anti-IL-17A reactivity Procarta commercial FMIA: IL-1β, IL-4, IL-6, IL-8, rPoIL-17F stimulated these prior to hybridoma fusion (July 2010) g a b 15000 IL-10, IFN- , TNF- , TGF- cells to express both IL-6 rPoIL-17A http://www.panomics.com/index.php?id=product_35 • Screen hybridomas for growth (July 2010; 47%); and IL-8 protein at 24 hours. 12000 rPoIL-17F • Test supernatants for reactivity with yeast proteins by The rPoIL-17A stimulated at 9000 ELISA (Aug. 2010); but colonies stopped growing similar levels to E. coli

derived rHuIL-17A (R&D 6000 • New fusion screening underway (November, 2011) IL-8 [pg/ml] IL-8 Comparison Sensitivity and Specificity of CCL2 assays #317-ILB) at the same 3000 • Future plans: Once cloned, supernatants screened for concentration. reactivity with native mammalian protein, e.g., by ELISA, 0 pAb based VetSet ELISA Luminex assay for rPoCCL2 0.001 0.01 0.1 1 10 100 1000 Luminex, effect on bioactivity at BARC. SA CCL2 (74) IL-17 [ng/ml] • Test reactivity using flow cytometry based on intracellular staining of cells stimulated with Con A, IL-6 20000.00 Development of panel of Porcine and TGFb followed by PMA and ionomycin

10000.00

Fluorescence(FI) Intensity IFN-a and IFN-β reagents Goal: produce a Luminex assay for PoIFN-a and PoIFN-β. 0.00 Publications

10.00 100.00 1000.00 Recombinant proteins: Kingfisher yeast rPoIFN-a, rPoIFN-β Concentration (pg/ ml) Boyd P, Hudgens E, Loftus JP, Tompkins D, Wysocki M, Kakach LT, Labresh J, Baldwin CL, a UMass mammalian rPoIFN- and rPoIFN-β-His/Myc Lunney JK. 2010. Expressed gene sequence and bioactivity of the IFNgamma-response Native protein: supernatants from Poly I:C stimulated PK15 chemokine CXCL11 of swine and cattle. Vet Immunol Immunopathol. 136: 170-5. ELISA Luminex cells for PoIFN-β; from PHA stimulated PBMC for PoIFN-a Hudgens E, Tompkins D, Boyd P, Lunney JK, Horohov D, Baldwin CL. 2011. Expressed gene sample# Cells Culture pg/ml Specificity rPo Chemokines Hybridomas: sequence and bioactivity of the IFNγ-response chemokine CXCL9 of cattle, horses and 91050 PBMC 18 hr media 430 <10 10pg/ml ELISA Luminex swine. Vet. Immunol. Immunopathol. 141: 317-21. Anti-PoIFN-β: DT2, DT17, and DT24 from UMass VIRN with CCL3-10 Lawson S, Lunney J, Zuckermann F, Osorio F, Nelson E, Welbon C, Clement T, Fang Y, Wong 91051 PBMC 18 hr LPS [10ug/ml] 124174 230000 < 30 < 60 yeast protein; Strong binding but not specific. S, Kulas K, Christopher-Hennings J. 2010. Development of an 8-plex Luminex assay to PMA/Ion CCL4-10 91052 PBMC 18 hr [40ng/ml/1ug/ml] 125 <10 < 30 < 60 10E9, 1F8, 2E8 and 1G2 from A Garmendia at U CT against detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination. Vaccine. 28: 5356-64. CCL5-10 Adeno-rPoIFN-β construct; specificity under review 91053 PBMC 18 hr ConA [0.25ug/ml] 5805 6780 < 30 < 60 Wagner B, Hillegas J, Kabithe E, Boyd P, Zarlenga D, Zagorski B, Dawson H, Lunney, JK. Anti-PoIFN-a: Fusions screening underway at Umass; CCL20-10 2011. Development and characterization of a monoclonal antibody to porcine interleukin 4 91054 PBMC 18 hr PHA [0.5ug/ml] 33414 119900 < 30 < 60 New fusions aimed at rPoIFN-a1 vs rPoIFN-a6 using cloned receptor alpha (CD124). Vet. Immunol. Immunopathol. In Revision. Manuscripts on IL-13R, IL-17, CCL2, CCL3L1 + CCL4, CXCL9 are in preparation. 91055 PBMC 18 hr PWM [2ug/ml] 18375 123490 genes and peptides from Y Sang at Kansas State Univ. and fusions with B Wagner at Cornell Entrican G, Lunney JK. 2010. Veterinary Immunology Committee Toolkit Workshop 2010: KF#VS0081S-002; Lot# HJ0167GJ Lunney, Boyd, Hillegas, Freer, Crossman, Hudgens, Tompkins, Kakach, Sullivan, Progress and Plans. Vet. Immunol. Immunopathol. Submitted. LaBresh, Dawson, Baldwin, and Wagner. 2011. manuscript in preparation Other Plans Acknowledgements Major efforts have been taken to test whether anti-human (or other species) mAb Develop mAb to swine IgG isotypes cross react with swine proteins. Dr. Harry Dawson, BHNRC, BARC has performed Expression of chimeric camelid-swine IgG constructs for each swine IgG isotype, are being screens for numerous anti-porcine cell surface and internal antigens. To affirm these Supported by the USDA NIFA AFRI grants #2006-35204-16880 and developed with S. Muyldermans, Belgium, on a separate grant by J Butler, U Iowa using cross-reactions he has first had to establish appropriate screening assays, e.g., for renewal #2010-65121-20649 for the U.S. Veterinary Immune Reagent chimeric camelid-swine IgG constructs. IL-17A expression, and then affirm that unlabeled mAb blocks labeled mAb binding. Network, US DHS Interagency Agreement #HSHQDC10X00021, USDA As affirmed in the Dawson and Lunney labs a major issue is preparing enriched cell NIFA/DHS grant #2010-39559-21860 and by ARS project populations at the desired stage of activation for expression of the target antigen so Develop additional sensitive Luminex bead assays #1265-32000-098 funds. that the test of mAb reactivity can be performed. Multiplex bead (FMIA or Luminex) assays are being developed for other cytokines and Luminex assays were supported by National Pork Board grants #08-189 and chemokines using currently available or newly developed mAb reagents.

#09-244 Presented in Chicago IL USA at 2011 International Porcine Reproductive and Give your feedback please! The authors thank their Toolkit collaborators for their expertise and helpful Respiratory Syndrome Symposium (IPRRSS) Dec. 2-3, 2011 and the Conference of suggestions for these studies. Research Workers in Animal Diseases (CRWAD) meeting, Dec. 4-6, 2011