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
Research Effort Categories
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.
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).