DEVELOPMENT OF NEW SWINE REAGENTS TO BROADEN OUR UNDERSTANDING OF IMMUNE CORRELATES OF PROTECTION AND MICROBIAL PATHOGENESIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1019192
• Grant No.: 2019-67015-29815
• Project No.: OHO03045-CG
• Proposal No.: 2018-06965
• Program Code: A1223
• Project Start Date: Jul 15, 2019
• Project End Date: Jul 14, 2023
• Grant Year: 2019

Project Director
Gourapura, R. J.

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
OARDC Food Animal Health

Non Technical Summary
Pigs are a major food animal species in the US and globally. Losses caused by infectious diseases [e.g., porcine reproductive and respiratory syndrome, swine influenza and porcine epidemic diarrhea] demonstrate the need for effective vaccines, biotherapeutics and nutritional interventions. The pig is also an important biomedical model for development, obesity, cardiovascular, transplantation, and vaccine research. Current research efforts require a broad range of immune reagents, but those available for pigs are limited. Our goal is to generate these priority reagents and pipeline them for marketing. The project team will prioritize research targets based on international input from researchers. Newly identified immune pathways, updated listing of commercially available reagents, and open communication with peers will determine targets and avoid duplication. Based on our research expertise, and the experience gained from the past swine toolkit efforts, the team has identified best immunization and screening strategies, developed plans to collaborate with commercial partners for protein expression and monoclonal antibody (mAb) production, and updated protocols to efficiently evaluate specificity and utility of reagents. Our specific objectives are: 1) Clone and express important swine immune molecules; 2) Prepare panels of mAbs reactive with the swine immune targets and SLA class I tetramers; 3) Use reagents produced to develop new assays for swine immune markers; and 4) Provide the veterinary community with new commercial reagents and up-to-date information and techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine and biomedical research efforts substantially.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

0%

Developmental

100%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3599	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1090 - Immunology;

Keywords

swine

cytokines

immune reagents

mab

tetramers

Goals / Objectives
Our goal is to generate the priority reagents and pipeline them for marketing as affirmed by our Specific Objectives: 1) Clone and express important swine immune molecules; 2) Prepare panels of mAbs reactive with the swine immune targets, and SLA class I tetramers; 3) Use reagents produced to develop new assays for swine immune markers; and 4) Provide the veterinary community with new commercial reagents and up-to-date information and techniques for their research efforts.The project team will prioritize research targets based on international input from researchers, newly identified immune pathways, updated listing of commercially available reagents and open communication with peers to determine targets and avoid duplication of international toolkit efforts. Based on our combined research expertise, experience gained from the US VIRN and our ongoing US-UK collaborative activity, the team has identified improved immunization and screening strategies, collaborated with commercial partners for protein expression, mAb production and assay development, and updated protocols to efficiently evaluate specificity and utility of reagents. Because of the improved availability of swine immune reagents, this renewal application will focus on new targets (Th immune regulators and tetramers) while continuing to develop mAbs to important immune proteins. Tools and reagents generated by this project will undoubtedly advance swine and biomedical research efforts substantially.

Project Methods
Objective 1. Clone and express swine immune proteins For Obj.1, Kingfisher Biotech, Inc., will clone selected swine cytokines and immunological markers using their yeast Pichia pastoris expression system. Their team has enhanced their yeast expression system, using codon optimization to improve protein expression, different electroporation and growth conditions, as well as improved protein purification options. Kingfisher will provide the purified proteins, at cost to the project, for immunizations and screenings; they will also provide expressed proteins for other species to test for mAbs that recognize orthologous proteins. Simultaneously, Kingfisher will offer them to the community as commercial products. So, we will be quickly addressing our Obj.4 commercialization goal. Targets for mAbs to porcine immune proteins are planned; most are already available in µg quantities through Kingfisher Biotech. They will include IFNβ, IL-28B, IL-3, IL-5, IL-15, PD-L1, APRIL (TNFSF13A), BAFF (TNFSF13B) and TWEAK (TNFSF12). These targets were selected for their role in innate and adaptive immunity, regulatory T cells, antiviral and mucosal immunity. Most of these are already available as yeast expressed proteins at Kingfisher; so, if funded, we're ready to ship them for mAb production. Year 2 targets are expected to include IL-7 and IL-18, and other, more difficult to express proteins, including IL-29, IL-31 and IL-33 (if available). As noted in our Management Plan, most immunizations for cytokines and T and B cells factors will use yeast expressed proteins, and hybridoma development performed by commercial labs in the US. For secondary and functional screening, the positive mAbs will be provided to different coPIs labs for detailed specificity testing and assay development, as outlined below, and to swine immunologists for final verification. IFNβ is one of the important type I IFNs; it exhibits a variety of biological functions, including antiviral, anti-proliferative, immunomodulatory and developmental activities (92).T cell transcription factors: T-bet, GATA-3 and RORγt. These targets were chosen since they are critically involved in regulating the development, activation, and function of Th1, Th2, and Th17 cells, respectively, in multiple tissues including mucosal sites.The targets for anti-cell surface CD mAbs will include CD19, CTLA-4 ligands [sCD80 (B7-1) and sCD86 (B7-2)] and the continuation of the US UK grant effort for producing anti-CD1d mAbs. CD19 is a marker of all B cells, expressed on progenitor B cells, naïve and memory B cells, and plasmablasts. It is a transmembrane receptor that associates with CD21, CD81 and CD225 to alter B cell responses. An approach similar to that previously reported for human CD19 extracellular domain (ECD) will be followed for protein synthesis (98). It will be expressed, first in yeast, or in insect cells if necessary. and subsequently be used for immunizations. Past attempts using an E. coli expression system (JE Butler) and equine vector expression (B Wagner) (99) were unsuccessful. While there are antibodies to human C-terminal cytoplasmic tail, the utility is limited to western blotting. For CTLA-4 ligands, the external soluble portions [sCD80 (B7-1) and sCD86 (B7-2)] will be expressed at Kingfisher.Objective 2. Prepare panels of mAb reactive with swine immune targets; prepare SLA class I tetramers.For Obj.2, panels of mAbs for swine cytokines will be produced under contract with company 1 or an alternate source. This process can start immediately as there are already proteins in the process of expression (IL-3, IL-5, IL-15, IL-28B, PD-L1, BAFF, APRIL and TWEAK) at Kingfisher Biotech, Inc. for which no panel of mAbs are yet available. As shown in Table 1, each immunization and fusion is expected to result in a panel of ~50 hybridomas from which the 8-12 best hybridoma clones will be expanded for each target. These will enable the team to identify epitope reactivity, test for effects on bioassay, neutralization assays and cell staining, and expand the swine BBMAs (100, 101).Primary screening of the hybridoma supernatants: Yeast expressed proteins are highly glycosylated. Thus, all hybridoma supernatants will be dual screened: against the target antigen as well as a negative control yeast protein. We have chosen yeast expressed boIL-4 for the negative control since it is expressed well in yeast and is highly glycosylated, the most common epitope recognized by cross-reactive mAbs. The selected hybridomas will then be recloned, specificity reaffirmed. Once characterized, mAbs have been identified, hybridomas will be adapted to serum free (or low IgG) medium and a large amount of supernatant collected. The final supernatants will be provided to BARC for mAb purification using protein A column for further analyses. Aliquots of frozen hybridoma cells will be provided to OSU and BARC for long term storage.Secondary screening of T and B cell transcription and activation factors: Flow cytometry for secondary screening will involve differential cell screening for highly expressed ligands/factors PD-L1, BAFF, APRIL, RORγt, T-bet and GATA-3 by T and B cells, based on cell subset expression or up/down regulation after stimulus. Briefly, normal healthy pig cells from selected tissues or PBMCs stimulated overnight with appropriate stimulant, LPS or PMA/Ionomycin in the absence (for non-secretory proteins T-bet, GATA-3 and RORgt) or presence of monensin and brefeldin (for secretory proteins). The cells will be externally stained with fluorochrome labeled anti-CD163, CD172a, CD3, CD4 and CD8 mAbs, and then intracellularly stained with hybridoma supernatants, followed by appropriate isotype specific secondary fluorochrome conjugated anti-mIgG (or fluorophore labeled purified mAbs) to identify specific cell types expressing the target protein. At NADC, intestinal intraepithelial cells and lamina propria cells will be isolated and reactivity of RORgt, T-bet, and GATA-3 tested in conjunction with CD45, CD3 and CD21 to identify innate lymphoid cells (ILCs). Flow cytometric analyses will be performed to determine the frequency of transcription and activation factors and CD+ cells among various subsets of pig cells by acquiring 100,000 events in a flow cytometer and analyzed using the FlowJo software. Suitable isotype control mAbs will also be included. The frequency of target CD expressing cells in double and triple positive cells will be determined. Positive hybridoma supernatants could also be screened for their applicability in immunoblots (native and denaturing gels) and immunofluorescence of fixed samples.Production of soluble porcine SLA class I (pSLA-I) for tetramers. To maximize efficiency and provide maximal resources to the research community, we will focus on producing SLA tetramers for three different SLA alleles in which IAV specific epitopes have already been identified (97) and that are known to be in high frequency in the U. S. swine herd (45). The synthesis and validation of multiple SLA tetramers allows for future adaptation to additional swine pathogens, as opposed to testing multiple IAV peptides with only a single SLA. In addition, we have selected peptides conserved across multiple subtypes (H1 and H3) and clades within each subtype, giving IAV researchers maximal flexibility in their studies (i.e., not limited to using a single strain of IAV to utilize the tetramers) (97). Specially, SLA-1*0401, SLA-1*0101 and SLA-2*0401 will be synthesized. SLA-2*0401 and SLA-1*0401 can be folded with peptide NP-28 (CTELKLSDY), and SLA-1*0401 and SLA-1*0101 can be folded with M-20 (LTEVETYVL). The pairing of these SLAs with specific peptides has either been reported as a specific ligand (47) or validated with IFNg recall assays (97).

Progress 07/15/19 to 07/14/23

Outputs
Target Audience:Stakeholders, researchers, students and postdocs Changes/Problems:Our tasked research aim was to produce soluble porcine swine leukocyte antigen (SLA) for creation of SLA tetramers for three different SLA alleles in which influenza A virus specific epitopes. However, we were not successful in our efforts in tetramer production. Similarly, generation of new panels of mAbs reactive with porcine IL-5 and IL-21 has not been successful despite multiple hybridoma fusion attempts. Several studies are still ongoing due to lack of supplied kits from BD Bioscience. What opportunities for training and professional development has the project provided?Jovan Aquino, Johns Hopkins MS Biotechnology (2020-23) Milan Royer, Purdue Univ. Animal Science undergraduate (2023) Visiting PhD Scientist, Chaohui Dai, Yangzhou University, Yangzhou, Jiangsu, China (2020-21) Raksha Suresh is a MS student worked on immunohistochemistry How have the results been disseminated to communities of interest?Presented the posters in the below meetings attended by immunologists CRWAD meeting, Chicago IL, Jan 2023. IMMUNOLOGY2023, Washington DC, May 2023. Symposium in Honor of Bill Davis: Applications of Monoclonal Antibodies, May 2023, Pullman, Washington. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The USDA-NIFA Swine Immune Toolkit Initiative's goal has been to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our accomplishments corresponding to Specific Objectives: Clone and express important swine immune molecules and 2) Prepare panels of mAbs reactive with the swine immune targets, and SLA class I tetramers. We expressed soluble proteins and produced panels of monoclonal antibodies (mAbs) with the help of collaborations with commercial partners for protein expression and mAb production. Once purified, large batches of mAbs were made available to screen for their epitope specificity and reactivity in multiple immune assays. For each target a contractor had produced a panel of 9-11 mAbs and purified them. We tested whether any of the mAbs had reacted with orthologous proteins and searched for epitopes using that data and cross inhibition studies. Panels of purified mAbs to IL-6, IL-13, CXCL10, IL-17A, IL-28B, IFNg, CXCL10, and BAFF were generated and screened for epitope reactivity. 3) Use reagents produced to develop new assays for swine immune markers Based on those assignments made in objective 2, mAbs representing each epitope were labeled with AF647 for intracellular staining. For certain pig immune proteins our analyses showed that PMA/ionomycin stimulation of peripheral blood mononuclear cells (PBMC) stimulated intracellular expression.Panels of purified mAbs to IL-6, IL-13, CXCL10, IL-17A, IL-28B, IFNg, CXCL10, and BAFF were fluorescently labeled for screening for intracellular staining of porcine cells, including activated myeloid and T cells, and cell lines such as IPEC-J2, using flow cytometry-based assays. Intracellular staining results have been confirmed for porcine IL-17A on PMA/Ionomycin stimulated cells (Manirarora et al 2022). IFNg stimulated cells were shown to express CXCL10. These cells were further phenotyped by triple staining for CD3, CD4, CD172 and CXCL10 with the result that CD172+ monocytes were consistently CXCL10+ with a subset of CD4+ cells under some conditions. Comparable CXCL10 induction in these immune cells determined that anti-PoCXCL10-1.4 mAb is the best mAb clone for intracellular staining (Hailstock et al, Manuscript in preparation). Our panel of mAbs were used to develop quantitative sandwich ELISAs. Now sensitive sandwich ELISAs are available for IL-17A, IL-13, and CXCL10; other targets are being screened for best mAb pairs for such assays. Immunohistochemistry analyses for binding of two of the anti-PoCXCL10 mAb on formalin fixed pig lymph nodes and spleen tissues was confirmed successfully. CD3 antibody for T cell activation - ThePPT7clone of anti-PoCD3 antibody was received from Vienna for testing its efficacy in pig T cell activation. As previously shown, the PPT7 clone activated pig T cells to proliferate and produce IFN-gamma. The PPT7 hybridoma intellectual property is held by UK Pirbright Institute, and they have sequenced the immunoglobulin gene of the PPT7 clone held at Pirbright, and it is identical to the clone in Vienna. Pirbright is currently working with Bio-Rad (formerly AbD Serotech) to commercialize the PPT7 clone. Samples were provided to Co-PIs on the proposal for testing of reagents, including blood for PBMC and lung lavage for macrophages. Dr. Loving's group has kept contact with European Reagent Group to track their progress on an anti-CD19 antibody for pigs. If they succeed in developing the reagent, resources will be redirected to other targets. Reagents were procured from a company to test all of their anti-human and anti-mouse surface marker antibodies for reactivity to porcine cells, which includes more than 400 reagents. 4) Provide the veterinary community with new commercial reagents and up-to-date information and techniques for their research efforts The immunological techniques and tools will be useful for evaluating future swine infectious disease immunity and vaccine responses. We provided these reagents to many collaborators to affirm reactivities and have sent panels of mAbs to colleagues in Czech, Denmark, Germany and Canada. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Patil, V, J.F Hernandez-Franco, G. Yadagiri, D. Bugybayeva, S. Dolatyabi, N. Feliciano-Ruiz, J. Schrock, J. Hanson, J. Ngunjiri, H. HogenEsch and G.J. Renukaradhya* (2022). A split influenza vaccine formulated with a combination adjuvant composed of alpha-D-glucan nanoparticles and a STING agonist elicits cross-protective immunity in pigs. Journal of Nanobiotechnology 20(1):477. PMID: 36369044, PMCID: PMC9652892. 2. do Nascimento, G.M, D. Bugybayeva, V. Patil, J. Schrock, G. Yadaigiri, G.J. Renukaradhya and Diel, D.G (2023). An Orf-Virus (ORFV)-Based Vector Expressing a Consensus H1 Hemagglutinin Provides Protection against Diverse Swine Influenza Viruses. Viruses, 15(4):994. PMID: 37112974

Progress 07/15/21 to 07/14/22

Outputs
Target Audience:Swine immunolgy researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Student training: S Chick was a USDA Pathways student for this year. She graduated with a B.S. in Animal Science from VA Tech in Dec. 2021. She's starting as a PhD candidate in Dairy Science at VA Tech, Blacksburg, VA in August 2022. Jovan Aquino was an HBCU Pathways student who completed his M.S. in Biotechnology from Johns Hopkins Univ. in May 2022. He is a USDA ARS APDL technician working part-time on this project. How have the results been disseminated to communities of interest?Presented the posters in the below meetigns attended by immunolgisits CRWAD meeting, Chicago IL, Dec 2021. IMMUNOLOGY2022, Portland, Oregon, May 2022. Swine in Biomedical Research Conference 2022, Madison, Wisconsin, June 2022. What do you plan to do during the next reporting period to accomplish the goals?Work is underway to test the mAb panels forintracellular stainingof porcine immune cells using flow cytometry if appropriate stimulation conditions were identified, including immune cells isolated from vaccinated and influenza virus challenged pigs.

Impacts
What was accomplished under these goals? Our team concentrated on characterizing panels of mAbs immune proteins, the interleukins (IL), interferons (IFN) and chemokines. We collaborated with our commercial partner, Kingfisher Biotech, to express the porcine proteins using their yeast expression system. For our toolkit efforts we use the recombinant proteins for production of panels of monoclonal antibodies (mAbs). For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques using these reagents for their research efforts. In FY22 we focused our work on panels of mAbs reactive to porcine CXCL10, BAFF, IL-5, IL-13, IL-17A, IL-21, IFNb, and IL-28B. We tested the mAbs for reactivity on protein orthologues from other species. We aimed to affirm that the mAbs react with the native porcine protein, which has been successful for several targets but not for IFNb. Intracellular staining results have been confirmed for porcine IL-17A and CXCL10 mAbs. Sensitive sandwich ELISAs are available for IL-17A, IL-13, and CXCL10; other targets are being screened for best mAb pairs for such assays. We welcome collaborators to affirm reactivities and have recently sent panels of mAbs to colleagues in Czech. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Mair KH, Crossman AJ, Wagner B, Babasyan S, Noronha L, Boyd P, Zarlenga D, Stadler M. van Dongen KA, Gerner W, Saalmueller A, Lunney JK. 2022. The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) is expressed on the majority of porcine NK cells ex vivo without stimulation. Frontiers Immunol. 13:767530. doi: 10.3389/fimmu.2022.767530.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Manirarora J, Walker K, Patil V, Renukaradhya G, LaBresh J, Sullivan Y, Francis O, Lunney JK. 2022. Development and characterization of new monoclonal antibodies against porcine Interleukin -17A and Interferon-gamma. Frontiers Immunol. Comparative Immunology. 13:786396. doi: 10.3389/fimmu.2022.786396
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: GJ Renukaradhya, C Loving, S Kenney, J LaBresh, V Patil, K Byrne, JN Manirarora, KE Walker, C Dai, T Hailstock, JK Lunney. Development of swine immune reagents for analysis of immune correlates for vaccine, infection, and in biomedical research. CRWAD, Chicago IL, Dec 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: T Hailstock, C Dai, J Aquino, KE Walker, JN Manirarora, GJ Renukaradhya, J LaBresh, JK Lunney. Characterization of anti-porcine CXCL10 monoclonal antibodies. CRWAD, Chicago IL, Dec 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: JK Lunney, T Hailstock, C Dai, J Aquino, S Chick, KE Walker, JN Manirarora, GJ Renukaradhya, Y Sullivan, J LaBresh. Characterization of anti-porcine CXCL10 monoclonal antibodies. IMMUNOLOGY2022, Portland, Oregon, May 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: GJ Renukaradhya, C Loving, S Kenney, J LaBresh, V Patil, K Byrne, JN Manirarora, KE Walker, J Aquino, S Chick, C Dai, T Hailstock, JK Lunney. Development of swine immune reagents for analysis of immune correlates for vaccine, infection, and in biomedical research. IMMUNOLOGY2022, Portland, Oregon, May 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: JK Lunney, J Aquino, KE Walker, S Chick, C Loving, J LaBresh, S Kenney, GJ Renukaradhya. Current research for developing swine immune reagents. Swine in Biomedical Research Conference 2022, Madison, Wisconsin, June 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: JK Lunney. Progress and Challenges in developing swine immune reagents. Swine in Biomedical Research Conference 2022, Madison, Wisconsin, June 2022.

Progress 07/15/20 to 07/14/21

Outputs
Target Audience:Stakeholders, students and postdocs Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Student and Trainees training: T Hailstockwas an HBCU Pathways studentfor the first 2 years of this project. She's now a PhD candidate at Emory Univ, Atlanta GA Chaohui Dai was a 1 year exchange student supported by Yangzhou University International Academic Exchange Fund YZUIAEF201901005. Jovan Aquino was an HBCU Pathways student who is now an APDL technician working part-time on this project. Veerupaxagouda Patil, a research associate is working part time on this project How have the results been disseminated to communities of interest?Renukaradhya, G.J, C Loving, S Kenney, J LaBresh, V Patil, K Byrne, K Walker, C Dai, T Hailstock, JK Lunney. Development and characterization of new swine immune reagents to understand immune correlates for vaccines, infection, and biomedical research outcomes. 101st Annual Conference of Research Workers in Animal Diseases Virtual meeting, December 5-8, 2020. Renukaradhya, G.J,C Loving, S Kenney, J LaBresh, V Patil, K Byrne, K Walker, C Dai,T Hailstock, JK Lunney.Development and characterization of novelswine immune reagents to understand immune correlates for vaccines, infections, and biomedical research outcomes.Virtual IMMUNOLOGY2021™,May 10-15, 2021. What do you plan to do during the next reporting period to accomplish the goals?The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts will be aimed at chareacterization of anti swine IL-5 and IL-21 mAbs generated through this grant funding.

Impacts
What was accomplished under these goals? The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts are aimed at expression of soluble proteins and production of panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. Our team concentrated on characterizing panels of mAbs anti-IL-17A, IFNg and CXCL10. For each target a contractor had produced a panel of 9-11 mAbs and purified them. Previously we had tested whether any of the mAbs had reacted with orthologous proteins and searched for epitopes using that data and cross inhibition studies. Based on those assignments mAbs representing each epitope were labeled with AF647 so intracellular staining could be evaluated. Our analyses showed that PMA/ionomycin stimulation of peripheral blood mononuclear cells (PBMC) induced significant expression of IL-17A by CD3+ T cells (1.01-1.19%double positive cells).These were consistently higher when compared to a cross reactive anti-human mAb (0.69% double positive cells). This mayindicate that the anti-porcine mAbs will detect a broader range of IL-17A+ cells. We plan to affirm that with further in vivo studies.For the anti-IFNg mAbs no consistent intracellular staining was detected. Our panel of 10 aCXCL10 mAbs was used to develop a quantitative sandwich ELISA, with verified reactivity with both the yeast expressed rPoCXCL10 as well as the native porcine CXCL10. Select anti-CXCL10 mAbs from this panel were screened for intracellular staining of PBMCs and spleen cells. Naive and PMA/Ionomycin or IFNg stimulated cells were shown to express CXCL10. These cells were further phenotyped by triple staining for CD3, CD4, CD172 and CXCL10 with the result that CD172+ monocytes were consistently CXCL10+ with a subset of CD4+ cells under some conditions. Comparable CXCL10 induction in these immune cells determined that aCXCL10-1.4 mAb is the best mAb clone for intracellular staining. These techniques and tools will be useful for evaluating future swine infectious disease immunity and vaccine responses. Generation of new panels of mAbs reactive with porcine IL-5 and IL-21 has been partially successful with generation of IL-5 (5 potential) and IL-21 (only 2) clones with the help of commercial partners. Future work on these clones on their characterization is in progress. Planning for the generation of SLA-I & -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides has been initiated. Panels of immune reagents are required to perform complex immune studies; those currently available for pigs are limited. For each target, our goal is to provide the veterinary community with the new commercial reagents and standardized techniques in using these new reagents for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts. Samples were provided for testing of reagents, including blood for PBMC and lung lavage for macrophages to Co-PIs on the proposal. Dr. Loving's group has kept contact with European Reagent Group to track their progress on an anti-CD19 antibody for pigs. If they succeed in developing the reagent, resources will be redirected to other targets.Reagents were procured from a company to test all of their anti-human and anti-mouse surface marker antibodies for reactivity to porcine cells, which includes more than 400 reagents. Our tasked research aim is to produce soluble porcine swine leukocyte antigen (SLA) for creation of SLA tetramers for three different SLA alleles in which IAV specific epitopes have already been identified. We are in the initial stages of planning and executing tetramer production. Attempts to acquire porcine beta 2 microglobulin and SLA biotinylation expression constructs (essential for tetramer folding) from Emory University have run into continued problems obtaining signed material transfer agreements. We have instead chosen to create our own beta 2 microglobulin and SLA biotinylation expression constructs. Utilizing https://www.ebi.ac.uk/ipd/mhc/group/SLA we have identified three of the most commonly expressed SLA alleles in commercial swine breed backgrounds: SLA-1*0101, SLA1*04:01:01, and SLA-2*0401. Synthetic DNA constructs encoding porcine beta 2 microglobulin and the specified SLAs have been designed and order from Integrated DNA Technologies. Once COVID-19 related research restrictions at OSU are lifted these DNAs will be cloned into bacterial and eukaryotic expression systems and protein production followed by tetramer folding will be attempted.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Intranasal Delivery of Inactivated Influenza Virus and Poly(I:C) Adsorbed Corn-Based Nanoparticle Vaccine Elicited Robust Antigen-Specific Cell-Mediated Immune Responses in Maternal Antibody Positive Nursery Pigs. Patil V, Renu S, Feliciano-Ruiz N, Han Y, Ramesh A, Schrock J, Dhakal S, HogenEsch H, Renukaradhya GJ.Front Immunol. 2020 Dec 16;11:596964. doi: 10.3389/fimmu.2020.596964. Porcine cytokines, chemokines and growth factors: 2019 update. Dawson HD, Sang Y, Lunney JK.Res Vet Sci. 2020 Aug;131:266-300. doi: 10.1016/j.rvsc.2020.04.022. Epub 2020 May 4.PMID: 32442727 Review. Neonatal and infant immunity for tuberculosis vaccine development: importance of age-matched animal models. Ramos L, Lunney JK, Gonzalez-Juarrero M.Dis Model Mech. 2020 Sep 15;13(9):dmm045740. doi: 10.1242/dmm.045740.

Progress 07/15/19 to 07/14/20

Outputs
Target Audience:Stakeholders, students and postdocs Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Organized aworkshop on swine immune toolkitin the International Veterinary Immunology Symposium in August 2019, Seattle, Washington What do you plan to do during the next reporting period to accomplish the goals?The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts will be aimed at expression of soluble proteins and production of panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production, and their charecterization.

Impacts
What was accomplished under these goals? Generation of new panels of mAbs reactive with porcine IL-5 and IL-21 has been initiated with the help of commercial partners.Hybridomas expressing new panels of 9-10 mAbs reactive with porcine BAFF and IL-28B are now being expanded under contract with our commercial partner. Once purified at BARC large batches of mAbs will be available to screen for their epitope specificity and reactivity in multiple immune assays. Panels of purified mAbs to IL-6, IL-13, and CXCL10, that were previously been screened for epitope reactivity, will be fluorescently labeled and screened for intracellular staining of porcine cells, including activated myeloid and T cells, and cell lines such as IPEC-J2, using flow cytometry-based assays. A sensitive sandwich ELISA assay was developed for IL-17A protein quantitation. MAb reagents to IL-13 and IL-6 will now be screened for best mAb pairs for such assays. CD3 antibody for T cell activation - The PPT7 clone of anti-CD3 antibody was received from Vienna for testing pig T cell activation. As previously shown, the PPT7 clone activated pig T cells to proliferate and produce IFN-gamma. The PPT7 hybridoma intellectual property is held by Pirbright, and they have sequenced the immunoglobulin gene of the PPT7 clone held at Pirbright, and it is identical to the clone in Vienna. Pirbright is currently working with Bio-Rad (formerly AbD Serotech) to commercialize the PPT7 clone. To support ongoing efforts towards development of antibodies to IL-6, supernatants from monocytes stimulated with lipopolysaccharide were provided to OSU-Gourapura and USDA-Lunney. Our tasked research aim is to produce soluble porcine swine leukocyte antigen (SLA) for creation of SLA tetramers for three different SLA alleles in which influenza A virus specific epitopes have already been identified. We are in the initial stages of planning and executing tetramer production. Attempts to acquire porcine beta 2 microglobulin and SLA biotinylation expression constructs (essential for tetramer folding) from Emory University have run into continued problems obtaining signed material transfer agreements. We have instead chosen to create our own beta 2 microglobulin and SLA biotinylation expression constructs. Utilizing https://www.ebi.ac.uk/ipd/mhc/group/SLA we have identified three of the most commonly expressed SLA alleles in commercial swine breed backgrounds: SLA-1*0101, SLA1*04:01:01, and SLA-2*0401. Synthetic DNA constructs encoding porcine beta 2 microglobulin and the specified SLAs have been designed and order from Integrated DNA Technologies. Once COVID-19 related research restrictions at OSU are lifted these DNAs will be cloned into bacterial and eukaryotic expression systems and protein production followed by tetramer folding will be attempted.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Entrican G, Lunney JK, Wattegedera SR, Mwangi W, Hope J, Hammond JA. 2020. The Veterinary Immunological Toolbox: Past, Present and Future. Frontiers Immunol., Comparative Immunol. In Press. 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