Source: AGRICULTURAL RESEARCH SERVICE submitted to
DEVELOPMENT OF POULTRY IMMUNE REAGENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012306
Grant No.
2017-67015-26793
Project No.
MD.W-2016-09335
Proposal No.
2016-09335
Multistate No.
(N/A)
Program Code
A1223
Project Start Date
Jun 15, 2017
Project End Date
Jun 14, 2022
Grant Year
2017
Project Director
Lillehoj, H.
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351
Performing Department
ARS/USDA
Non Technical Summary
This grant proposal presents a systematic plan and approach to continue to address the veterinary immunological reagent gap in the USA and other countries. A major obstacle to advances in poultry immunology and disease control is the lack of necessary poultry immunological reagents. Reagents are necessary (i) to determine the host's response to infections and to develop treatments to prevent these infections, and (ii) to develop vaccines against infectious diseases to improve poultry health. In this proposal, we propose to address the needs of poultry (primarily chickens), an economically important agricultural animal species worldwide. With increasing demands for high quality protein from poultry, we need to make sure that treatment strategies are developed against poultry infectious diseases since illnesses slow down the efficacy of global poultry production. Currently, there are two main challenges in poultry disease control: 1) the lack of poultry-specific immunological reagents, since mammalian reagents do not recognize poultry proteins, and 2) the lack of methods to effectively measure host immune response to many poultry infectious diseases. Consequently, there are no commercially available methods to measure poultry immunity. Successful accomplishment of this proposal will help new knowledge discovery and a new sets of poultry immune reagents and detection methods that will have a significant global impact in society (i.e., agriculture, human healthcare, food safety).
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132201090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1090 - Immunology;
Goals / Objectives
Worldwide demand for poultry meat is expected to outstrip that of other meats in the next decade, as poultry is the most cost-efficient feed-to-meat converter. However, the ever-present threat of infectious diseases impedes the efficacy and sustainability of global poultry production. Currently, there are two main challenges in poultry disease control: 1) a lack of immunological reagents, as mammalian reagents do not cross-react with the same molecules in birds, and 2) a lack of sensitive assays to assess poultry immunity. The overall goal of this project is to improve poultry health and production by developing new, publically accessible poultry immune reagents. This proposal outlines the systematic collaborative approaches to expand the work of the previous veterinary immune reagent development efforts of AFRI (US-VIRN) and is urgently needed to develop the immune reagents that are needed by the international poultry research community. We will canvass the avian immunology communities to identify 25 priority targets. The reagents produced will include bioactive cytokine/chemokine proteins and new cell surface markers, expressed using mammalian cells, Pichia pastoris or E. coli systems as well as antibodies to them. Our goal is to produce antibodies that function in ELISAs, for intracellular staining, for blocking function and signaling, and that are useful in flow cytometric and immunohistological applications. Successful completion of this proposal will dramatically speed the commercialization of poultry immune reagents, where it will have a significant global impact in multiple sectors (i.e. agriculture, food safety).Immune Reagent Grant is to continue to expand the repertoire of poultry immune reagents, with the following specific objectives: (i) to identify chicken immune molecules, particularly cytokines, chemokines and cell surface markers, express them as recombinant proteins, and characterize their function, (ii) to develop mAbs to the target chicken molecules, and (iii) to use these newly developed poultry immune reagents for multiplexed detection of inflammatory cytokine/chemokine levels in sera and tissues of infected and vaccinated birds.
Project Methods
Methods to clone and express chicken cytokine, chemokine, and CD genes and to purify their recombinant proteins are well-established in the PDs' laboratories. In all expression systems, the recombinant proteins will be tagged with either a His(6X) epitope for purification on a nickel affinity column, or a maltose-binding protein for purification on a maltose affinity column, as we described. E. coli expression will be carried out at ARS in the PDs' laboratories and yeast expression will be done by our collaborators at Kingfisher Biotech using a well-established P. pastoris expression system. The ARS lab (HL) will also express epitope-tagged recombinant proteins in mammalian cells, either transiently in COS-7 cells or stably in CHO cells using our established methods. Bioassays will be conducted in both PDs' laboratories to validate recombinant protein function. If necessary, bioassays for the equivalent molecule(s) in mammals will be adapted to the avian system. For chemotaxis assays, we will first isolate the appropriate cell type for the chemokine to be evaluated (e.g. heterophils for CXCLi2, B lymphocytes for CXCL13Li1, and monocytes for CCLi1 or CCLi2) and culture them in Boyden chambers to measure directed chemotaxis towards the chemokine gradient by cell counting or loading the cells with a dye and evaluating the dye concentration. For unknown CD genes, full-length cDNA sequences of the homologous genes from other species will be aligned and the regions of the gene with the greatest sequence identities will be used for primer design. If the PCR strategy fails, degenerate primers will be used to obtain the appropriate PCR product for DNA sequence analysis. Once recombinant CD proteins are expressed using the systems described above, mAbs will be produced using SOP. CD-specific mAbs will be screened using established flow cytometric methods with various lymphoid tissues and established chicken cell lines. Tissue reactivity will be validated by immunohistological techniques, as we described. We will generate antibodies against both existing recombinant proteins and proteins newly expressed in this grant .Mouse mAbs to new proteins will be produced by standard techniques. The mAbs will be screened by ELISA using the recombinant protein used for immunization or tested directly on appropriate cells by flow cytometry. They will be isotyped and tested in pairwise combinations for their ability to recognize different epitopes of the same molecule. All mAbs will be purified and one mAb of an antibody pair will be directly conjugated to an Alexa-Fluor fluorescent marker.All mAbs will be characterized in a number of standard assays using routine techniques. Firstly, as described above, the mAbs will have been screened either by ELISA against the immunizing recombinant protein or by flow cytometry against cells expressing the target molecule on their surface. The mAbs will also be tested by Western Blot and, if appropriate, for their ability to block the activity of the protein against which they were generated in a relevant bioassay. For immunohistochemistry, caecal tonsils, bursa of Fabricius, and spleen will be harvested from 3-week-old Specific Pathogen Free Avian Supplies (SPAFAS) chickens (Charles River Laboratories) and immediately frozen in liquid nitrogen. Cryostat tissue sections will be incubated with cytokine mAbs at 4°C for 18 hr, followed by biotinylated goat anti-mouse IgG secondary antibody (Vector, Burlingame, CA), the avidin-biotin-peroxidase complex (Vector), and 3-amino-9-ethylcarbazole substrate. Tissue sections will be observed by light microscopy at 400× magnification. To evaluate cells for intracellular cytokines using the mAbs, a general standard protocol available on the US-VIRN website (www.umass.edu/vetimm) will be used. Briefly, cultured chicken cells are stimulated for 24-96 hr with recombinant cytokines or mitogens to induce production of the cytokine of interest, and are restimulated during the last 4 hr of culture by addition of phorbol myristate acetate (PMA), ionomycin (0.5 mg/ml each), and monensin (2 mM) to facilitate intracellular cytokine accumulation. The cells are washed, fixed with 1% paraformaldehyde for 10 min at 22°C, and permeabilized with 0.1% saponin, 20% horse serum, and 0.1% sodium azide in PBS. Cells are reacted with the anti-cytokine mAb in 0.1% saponin, 5% horse serum, and 0.1% sodium azide for 45 min at 4°C, washed twice with 0.1% saponin, reacted with phycoerythrin-conjugated goat anti-mouse secondary antibody, and analyzed by flow cytometry for intracellular staining. Isotype-matched nonimmune IgG or IgM is used as a control for nonspecific staining. Kingfisher Biotech is creating ELISA VetSets for measuring cytokines and chemokines in cell culture supernatants or clinical samples, e.g., the chicken IL-16 set (www.kingfisherbiotech.com). Selection of the mAb pair(s) that produce the highest signal, minimal background, and "sandwich" the target antigen will be selected for further screening. Prior to final selection, mAb detection of natural samples will be confirmed and the ideal capture mAb concentration determined. These sets will contain ready-for-use, 96-well strip plates pre-coated with the optimized capture mAb. Selected mAbs pairs will also be evaluated for their suitability in Luminex™ multiplexed assays . Once the optimal antibody pair is selected, the ideal capture antibody concentration will be determined followed by determination of the detection antibody concentration. One of the available multiplex systems for protein quantification is the Luminex™ technology which is based on fluorescent beads that are color-coded into 100 distinct sets). To quantitatively measure cytokines and chemokines, each bead set will be coated with an antibody specific to a particular cytokine, allowing its capture from the sample. A mixture of fluorescently-labeled cytokine-specific antibodies is added for detection. Monoclonal antibodies are preferred over pAbs because they have been shown to improve the analytical sensitivity of the assay. Within the Luminex analyzer, lasers excite the internal dyes that identify each microsphere particle, and also any fluorescent reporter dye captured during the assay. Multiple readings are made on each bead set that result in an individual fluorescent signal for each bead assay. Our goal is to use our cytokine mAbs to establish multiplex assays for the detection Th1, Th2, and Th17 cytokines. All mAbs will be screened to ensure specific detection of native proteins using recombinant cytokines and the selection of mAb pairs that produce the highest signal, lowest background, and correctly "sandwich" the target protein will be selected for further screening.

Progress 06/15/17 to 06/14/18

Outputs
Target Audience:The target audiences will be scientists from academia, private company and government, veterinarians or farmers in poultry farm as well as avian scientists who study poultry diseases and immunology. During this reporting period, our focus was primarily on the development of poultry immune reagents for basic and applied research. When we complete the developing our panel of immune reagents for immune phenotyping in poultry, we plan to generate commercially available antibodies, immunoassays or ELISA kits with commercial partners for applied research in poultry that will be useful for field laboratories. All basic information on poultry immunity from this grant and all pertinent data will be shared with national and international poultry research communities through original manuscripts and/or workshops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several summer interns from local universities and high school were hired to be trained in immunology and molecular technology. Two graduate-level visiting students from Korea and China were also trained under this NIFA grant proposal. Two ORISE-fellows at the post-doc level were also hired under this grant to be trained in poultry immunology. How have the results been disseminated to communities of interest?We have presented our results at national meetings and published new findings in peer-reviewed papers. In order to share our immune reagents supported by this grant with public, especially the immunologist and avian scientists, we are communicating with Southern Biotech for licensing these reagents. We sent out a couple of our products to verify the applications on various conditions. Once the verification is done, Office of Technology Transfer (OTT) in ARS will be contacting to transfer the immune reagent for commercialization. What do you plan to do during the next reporting period to accomplish the goals?Besides 20 targets dealt in first year, as we stated in our project proposal, we will be selecting as least 5 more targets to identify 25 priority targets in total that we promised in our 5-year project plan. We will keep continuing 1) to express recombinant proteins, 2) to develop mAbs against them, 3) to characterize developed mAbs, and 4) to study the function and application in several disease models as we develop new immune reagents for poultry in collaboration with scientists from academia, government and private industry nationally and internationally.

Impacts
What was accomplished under these goals? The long-term goal of our efforts to develop immunological tools was to address significant knowledge and reagent gaps specific for poultry species in the U.S and other countries since limited availability of immune reagents and information on host immune response hinder critical progress in disease and vaccine research in this economically important agricultural animal. New discovery from this grant will advance our fundamental immunological knowledge as well as a new sets of poultry immune reagents and detection methods that will have a significant global impact in scientific community and society. Looking at the big picture, successful accomplishment of this grant will enhance the safety of the nation's agriculture food supply and address key issues on food security and food safety. Objectives 1. to identify chicken immune molecules, particularly cytokines, chemokines and cell surface markers, express them as recombinant proteins, and characterize their function 2. to develop mAbs to the target chicken molecules Under objective 1 and 2, significant progress has been made in identifying novel critical immune genes, expression of recombinant chicken proteins, developing hybridomas and monoclonal antibodies (mAbs) and characterizing their function and specificity. Dr. Woohyun Kim was hired to lead this project as a co-investigator and works closely with the PD (Dr. Lillehoj) and Dr. Li (Co-PI). This first year report contains progress made since June, 2017 and includes the identification of 20 candidate poultry target genes for intracellular and extracellular markers which are associated with host immune response. Cloning of new chicken genes were carried out the number of sets of primers which were designed and synthesized to amplify based on the chicken genomic and mRNA sequence. The recombinant proteins were obtained by transformation into E. coli, transfection into mammalian cells, or expression in yeast (collaboration with Kingfisher Biotech scientists). All recombinant proteins expressed have met the quality standard for immunization in mice (protein purity: ≥85%) for hybridoma production. For functional characterization of the recombinant proteins, several assays have been conducted including ELISA, nitric oxide assay, cell proliferation assay, qPCR, immunohistochemistry and Western blot. a. Chicken CXCLi2 (CXCL8, IL-8) The coding region of chCXCLi2 gene was cloned and expressed in E. coli and yeast for the development of mAbs and functional assays, respectively. Chicken CXCLi2 expressed from yeast induced chemotaxis of PBMCs, proliferation of macrophages, and expression of alpha smooth muscle actin in embryonic fibroblasts. We screened and selected two mAbs (#97 and #100) based on their specificity in sandwich ELISA. Both mAbs were characterized using Western blot, ELISA, immunocytochemistry, and neutralization assay and the results validated their identity, e.g., inhibition of chemotaxis, proliferation, and wound healing (Kim et al., 2017). b. Chicken IL-4 and IL-13 The chIL-4 gene was cloned, expressed in yeast and used for functional assay using chicken cells. Major new information on chicken IL-4 includes: 1) chIL-4 induced alternative activation of macrophages (AAMs) with inhibition of LPS-induced NO production, 2) chIL-4 decreased iNOS expression, increase arginase activity, and 3) chIL-4 induced M2 markers expression. Two mAbs (#6D9G and #8GE10) were used in ELISA, Western blot, immunocytochemistry and flow cytometry for detailed characterization. In neutralization assay, both mAbs blocked the reduction of NO production induced by chIL-4 and restore the expression of AAMs markers or M2 polarization (Chaudhari et al., 2018, under review). ChIL-13 is another Th2 cells-related cytokine with overlapped functions with IL-4. The chicken IL-13 gene was cloned and expressed in E. coli and will be used to immunize the mice for hybridoma production. c. Chicken IL-12 and IL-23 The genes of chIL-12p35, chIL-12p40, and chIL-23 were cloned and bioactive heterodimeric chIL-12 and chIL-23 were successfully expressed in mammalian expression system. We confirm that recombinant chIL-12 and chIL-23 form heterodimeric chains in SDS-PAGE and Western blot. The bioassay for chIL-12 and chIL-23 are ongoing and plans are underway to investigate the role of chIL-12 or chIL-23 in several poultry diseases such as coccidiosis and necrotic enteritis. After initial screening, 5 mAbs each detecting IL-12p40 and IL-23p19 were identified based on binding activity in ELISA and mAbs development for IL-12p35 is ongoing. As IL-23 and IL-12 are in the same IL-12 cytokine family and share the p40 subunit to form heterodimeric protein, five mAbs against IL-23p19 were also developed to establish the detection system differentiating IL-12 and IL-23 with same capture antibody, IL-12p40 mAb which is the common subunit of IL-12 and IL-23. d. Chicken IL-10 ChIL-10 was cloned, its sequence validated and recombinant chIL-10 was expressed in yeast. It was shown that chIL-10 is associated with host immune response against necrotic enteritis, an economically important intestinal disease caused by Clostridium perfringens. Five mAbs against chIL-10 has been developed and its binding specificity validated in antigen-specific capture ELISA. Efforts are ongoing to quantify the protein expression in biological samples obtained from necrotic enteritis-afflicted chickens. e. Chicken IL-7 and IL-7 receptor The chIL-7 and its receptor (CD127) genes have been cloned, their sequences validated and recombinant proteins produced. Studies to investigate the role and signaling process involving IL-7 and its receptor are ongoing. Both target proteins were used to immunize mice and the development of mAbs is ongoing. f. Chicken IL-17F, IL-21 and IL-22 Recombinant proteins from these three chicken cytokines were expressed and hybridomas were developed to identify mAbs which are specific against them. Functional and molecular characterizations will follow soon. g. Chicken chemokines and others Three chicken C-C motif chemokine ligands which are CCL4, CCL5 and CCL20 were selected to produce recombinant proteins and the mAbs against them. All three chemokines were expressed in yeast and the immunization and development of process is ongoing. Additionally, IL-16, IFN-α, and TGF-β genes were cloned and their recombinant proteins have been produced for mice immunization. Additionally, the genes encoding both targets will be cloned and expressed for their validation and characterization. In summary, our first year progress report contains the progress of developing 20 targets of poultry immune reagents at various stages of mAb production with the completion of 2 immune reagents with 2 peer-reviewed manuscripts, 3 projects in recombinant protein expression, 10 projects in mAb development and 5 projects in protein characterization. The mAbs stated here represent new sets of immune reagents which are specie-specific for poultry. These important new immune reagents will facilitate the discovery phase of their roles in host immune response against various infectious diseases which will impact the development of novel vaccines and antibiotic alternative strategies against various infections. Most importantly, these new poultry-specific immune reagents are critically needed now to develop specie-specific immune assays that can be readily adapted for the field assessment of infectious disease status. 3. to use these newly developed poultry immune reagent for multiplexed detection of inflammatory cytokine/chemokine levels in sera and tissues of infected and vaccinated birds The detection of immunological markers in a multiplexed platform will be carried out after the completion of several mAbs panels that we need for immune phenotyping of host response. For first year of project, our main focus is on developing mAbs specific for chicken-specific immune molecules.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kim, W.H., Lillehoj, H.S., Lim, Y., Min, W., Sullivan, Y.B., Kakach, L., and LaBresh, J.W. (2017). Development and characterization of mouse monoclonal antibodies reactive with chicken CXCLi2. Developmental & Comparative Immunology 72, 3036.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Truong, A.D., Hoang, C.T., Hong, Y., Lee, J., Lee, K., Lillehoj, H.S., and Hong, Y.H. (2017). Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex. Molecular Immunology 92, 5467.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Truong, A.D., Hoang, C.T., Hong, Y., Lee, J., Lee, K., Lillehoj, H.S., and Hong, Y.H. (2018). Dataset on characterization of recombinant interleukin-23?, IL-12p40 and IL-23 complex protein, which activates JAK-STAT signaling pathway in chicken cell lines using immunocytochemical staining. Data in Brief 16, 799805.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fernandez, C.P., Afrin, F., Flores, R.A., Kim, W.H., Jeong, J., Kim, S., Chang, H.H., Lillehoj, H.S., and Min, W. (2017). Downregulation of inflammatory cytokines by berberine attenuates Riemerella anatipestifer infection in ducks. Developmental & Comparative Immunology 77, 121127.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lin, R.-Q., Lillehoj, H.S., Lee, S.K., Oh, S., Panebra, A., and Lillehoj, E.P. (2017). Vaccination with Eimeria tenella elongation factor-1? recombinant protein induces protective immunity against E. tenella and E. maxima infections. Veterinary Parasitology 243, 7984.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Afrin, F., Fernandez, C.P., Flores, R.A., Kim, W.H., Jeong, J., Chang, H.H., Kim, S., Lillehoj, H.S., and Min, W. (2018). Downregulation of common cytokine receptor ? chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer. Developmental & Comparative Immunology 81, 225234.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fernandez, C.P., Afrin, F., Flores, R.A., Kim, W.H., Jeong, J., Kim, S., Lillehoj, H.S., and Min, W. (2018). Identification of duck IL-4 and its inhibitory effect on IL-17A expression in R. anatipestifer-stimulated splenic lymphocytes. Molecular Immunology 95, 2029.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Rengaraj, D., Troung, A.D., Lillehoj, H.S., Han, J.Y., and Hong, Y.H. (2018) Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens. Asian-Australasian Journal of Animal Sciences, accepted https://doi.org/10.5713/ajas.17.0836
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Kim, W.H., and Lillehoj, H.S. (2018) Immunity, immunomodulation, and antibiotic alternatives to maximize the genetic potential of poultry for growth and disease response. Animal Feed Science & Technology, under review
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Chaudhari, A.A., Kim, W.H., and Lillehoj, H.S. (2018) Interleukin-4 (IL-4) regulates alternative activation of macrophages in chickens: a sequential study using novel and specific neutralizing monoclonal antibodies against chicken IL-4. Developmental & Comparative Immunology, under review
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Kim, W.H. and Lillehoj, H.S. Intestinal immune responses to chicken coccidiosis in the context of Th1 and Th17 responses. International Congress of Mucosal Immunology (ICMI) in Washington, D.C., U.S. (2017).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Kim, W.H. and Lillehoj, H.S. Recent Progress in Understanding Host Immune Response to Avian Coccidiosis: Th1 and Th17 responses. American Society of Parasitologists and International Coccidiosis Conference (ASP-ICC) in San Antonio, TX, U.S. (2017)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Kim, W.H., Yim, Y., and Lillehoj, H.S. Detection of chicken interleukin-23, a heterodimeric cytokine with monoclonal antibodies reacting p19 and p40 subunits. American Association of Immunologists meeting (AAI) in Austin, TX, U.S. (2018)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Chaudhari, A.A., Kim, W.H., and Lillehoj, H.S. Production and characterization of monoclonal antibodies specific for chicken interleukin-4. American Association of Immunologists meeting (AAI) in Austin, TX, U.S. (2018)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Grant, A., Kim, W.H., and Lillehoj, H.S. Characterization of the immune reagent chicken IL-16. American Association of Immunologists meeting (AAI) in Austin, TX, U.S. (2018)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Sun, Z., Zhao, H., Kim, W.H., Panebra, A., Lillehoj, H.S., Xianyu, Z., Gu, C., and Li, C. Development and Characterization of Chicken CD127-Specific Antibodies. American Association of Immunologists meeting (AAI) in Austin, TX, U.S. (2018)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Lee, Y., Kim, W.H., Lee, S., and Lillehoj, H.S. Induction of interleukin-10 expression in chicken intestinal epithelial cells stimulated with Clostridium perfringens. Poultry Science Association of Annual Meeting (PSA) in San Antonio, TX, U.S. (2018)