Location: Animal Parasitic Diseases Laboratory
2020 Annual Report
Objectives
Highly infectious diseases of pigs present exceptional challenges to producers. Novel approaches are required to maintain animal health and welfare, particularly as scientists work to develop alternatives to the use of antibiotics for pathogen control. This project will explore immune and genomic-based approaches for understanding host-pathogen interactions. Probing the genetic variations associated with infection, immune evasion, innate and adaptive immune responses, and disease susceptibility and resistance will lead to improved animal health and alternatives for disease control and vaccine design. The goal of this research project is to develop effective countermeasures for preventing and controlling important respiratory diseases of pigs, such as Porcine Reproductive and Respiratory Syndrome (PRRS).
New genetic and immune markers will help producers and animal health professionals to prevent and control swine viral diseases; they will provide basic data to use for design of alternate control and vaccine strategies, thus decreasing production costs and improving trade potential. As a result of this work, animal health companies will have alternatives for discovering biotherapeutics and vaccines for swine respiratory diseases; pig breeding companies will have new tools to identify disease-resistant stock. Overall this project will stimulate advances in pig health that may be of broad economic importance.
Objective 1. Develop immunologic tools to evaluate swine immunity, including using immunological tools to enhance our understanding of swine immune system development [C4, PS4B], and using immunological tools to inform the design of novel innate immune intervention strategies to treat respiratory diseases of swine. [C2, PS2B]
Objective 2. Elucidate host response associated with swine respiratory disease and protective immunity, including discovering genetic and biological determinants associated with swine respiratory disease susceptibility, tolerance, or resistance, and discovering genetic and biologic determinants associated with good responders to swine respiratory disease vaccines. [C4, PS4B]
Approach
Characterize swine immune proteins (cytokines, chemokines) and monoclonal antibodies (mAbs) to these proteins and their receptors, and to antigens that define swine cell subsets and activation markers (CD antigens). To speed progress on reagent development, collaborate with commercial partners for protein expression and mAb production. Coordinate ARS efforts with NIFA supported US UK Swine Toolkit progress. Once panels of mAbs reactive with swine targets are available, test them for specificity and identify epitope reactivities to help develop sandwich ELISAs and Bead Based Multiplex Assays (BBMA). Work with USDA ARS and NIFA leadership to establish a veterinary immune reagent repository for relevant hybridomas and cell lines from various livestock species as well as to provide an updated website www.vetimm.org highlighting the availability of these reagents.
Emerging and re-emerging infectious diseases heighten the need to use the expanded swine toolkit to facilitate veterinary and biomedical research. Complex immune interactions determine the efficacy of a pig's response to infection, vaccination and therapeutics. New tools developed through this project, and the US UK Swine Toolkit grant, will expand options for probing mechanisms involved in disease and vaccine responses. Continue to assess samples archived through the PRRS Host Genetics Consortium (PHGC) for protein and metabalome alterations that may be predictive of PRRS viral levels or weight gain changes at different time-points post infection. Expand efforts to use pig as an important biomedical model including tuberculosis (TB) research. For TB test whether vaccination in neonatal minipigs leads to the development of immune responses similar to those described in human infants. Results from these trials will allow study of infant TB and TB vaccine efficacy. address biomedical
Following up on PHGC studies, as part of a USDA NIFA translational genomics grant, a more complex model, testing vaccination for PRRS followed by PRRSV and porcine circovirus (PCV) challenge [a PCV associated disease (PCVAD) model] was pursued. This approaches typical farm conditions and enables us to ask about the effectiveness of vaccination prior to PRRSV and PCV2 challenge. Additionally, data was collected on genetically defined pigs in true field trial conditions, providing data that is essential for transfer (and affirmation) of our disease genetic results to pig breeders. We expect that the combined models and genomic approaches will lead to identification of chromosomal regions, putative candidate genes and mechanisms involved in regulating pig responses to viral infections, vaccinations, and associated growth effects.For this ARS project we will evaluate the effect of anti-viral response pathways and biomarkers on vaccine and infection responses.
We will use RNAseq analyses to provide a more complete picture and reveal details of regulatory mechanisms impacting pig responses to vaccination, viral infection, and differential growth effects. Our proposed studies will expand analyses of samples collected on the grant funded 4 vaccination/PCVAD trials and 6 field trials (Appendix). As we identify
Progress Report
Progress was made on both project objectives and their subobjectives, all of which fall under National Program 103, Animal Health. They address NP103 Component 4: Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases and NP103 Component 2: Antimicrobial Resistance, Problem Statement 2B: Alternatives to Antibiotics. All milestones were “Met” or “Substantially met” in FY2020.
We cloned and expressed several swine immune proteins (cytokines, chemokines, transcription factors), working with commercial partners. A second commercial partner developed monoclonal antibodies (mAbs) to some of these proteins. These included B-cell activating factor (BAFF) and interleukin-28 (IL-28). We characterized panels of mAbs to ILs and cytokines in collaboration with university partners. We then characterized sets for transfer to commercial partners. These essential reagents advance international pig health and vaccination research efforts.
We developed a comprehensive catalog for swine immune reagents. Swine immunologists require immune markers and reagents. ARS scientists at Beltsville, Maryland, therefore used the most recent swine genome build to identify and annotate 289 immune-regulating proteins, including cytokines, chemokines and growth factor proteins. Searching all relevant resources and literature, the team identified all known commercial reagents, and verified availability of expressed proteins for 170 pig cytokines, chemokines and growth factors. Of these, 118 are recognized by at least one antibody reagent, 66 have a cloned recombinant peptide, and 97 have a proven quantitative assay. These data will be used by researchers worldwide to enhance swine immune studies, improving swine health and supporting biomedical research benefitting human health.
In collaboration with UK partners, we added all swine data previously housed at www.vetimm.org to the new Pirbright/Roslin UK Immunological Toolbox website and database (http://www.immunologicaltoolbox.co.uk). In addition, we updated swine immune marker data using the newly refined swine genome build (Sus scrofa 11.1) and using information collected in an exhaustive search of internet and commercial resources. These efforts identified mAbs and polyclonal antibodies that react with porcine cytokines, chemokines, growth factors as well as cloned recombinant proteins and assays for their quantitation.
Our international team organized a compendium of swine leukocyte antigens. Genomics has revolutionized our understanding of the swine immune system. The swine major histocompatibility complex (SLA) regulates T cell immunity. It therefore determines the intensity of disease and vaccine responses. The SLA complex maps to swine chromosome 7 and encodes approximately 150 loci, at least 120 of which are presumed functional. ARS scientists at Beltsville, Maryland, worked with scientists from the USA (U.S.), Austria, Japan, and France to annotate all recognized SLA genes. To do so they used the newest Sus scrofa genome build. They updated SLA nomenclature, reviewed SLA genetic typing methods, defined SLA protein expression, and described their roles in antigen presentation and immune, disease, and vaccine responses. They updated the role of SLA genes in xenotransplantation (cross species transplantation). Overall, these data provide swine researchers essential information on SLA genes and proteins and underscore their importance in swine health, disease, and vaccine responses.
We continued efforts to improve analyses of porcine reproductive and respiratory syndrome virus (PRRSV) infection responses by profiling gene expression in blood. These efforts identified biomarkers of PRRS resistance and susceptibility. We proved that including a novel porcine globin blocker improves library preparation by reducing the representation of an otherwise abundant transcript. We streamlined RNAseq analyses of blood cell RNA by switching to QuantSeq 3'mRNA sequencing. We refined methods targeting gene expression using NanoString arrays, improving our ability to study porcine immune and anti-viral responses. Finally, we used advanced biostatistical analyses to more completely describe regulatory mechanisms influencing anti-PRRS immune responses.
We explored genetic and biological determinants of PRRSV infection in nursery and reproductive pig models. Expanding on the previously-discovered swine chromosome 4 (SSC4) genetic viral resistance allele, we tested the effects of naturally-occurring single nucleotide polymorphisms (SNP) in the PRRSV receptor, CD163, and in several other genes. If confirmed, these SNPs may aid efforts to breed pigs resistant to PRRSV infection. We used transcriptional studies to evaluate what controls the timing of virus transfer from mother to fetus and to determine how fetuses control congenital infection.
Congenital PRRSV severity predicted using placental and fetal gene expression. Porcine reproductive and respiratory syndrome virus (PRRSV) infections cause an estimated $300 million losses in the U.S., annually. A pregnant gilt infected with PRRSV can transmit the virus to her fetuses. Fetal outcomes vary widely, but we do not adequately understand why. ARS scientists in Beltsville, Maryland, therefore worked with scientists at the University of Saskatchewan to probe maternal and fetal factors that predict disease severity and fetal resilience. Twelve days after the mother was exposed, fetuses were grouped by preservation status and viral level (VL) in the fetal placenta, serum and thymus. They discovered that fetuses begin responding only after the onset of detectable viremia. They identified biomarkers of susceptibility that may partially explain fetal demise. The effects of VL were evident in the thymus. High VL occurred in fetuses expressing notably low levels of several protective innate and adaptive immune pathways. Overall, this work indicates that fetal responses are governed by activities in the fetus and at the maternal-fetal interface. The newly-discovered biomarkers may help breeders improve animal health. These markers also suggest new anti-viral therapeutic approaches.
Accomplishments
1. Piglets are an important model for developing tuberculosis vaccines for human babies. The only vaccine available against human tuberculosis (TB) is Bacillus Calmette-Guerin (BCG). Better vaccines are needed, especially for newborns. But human neonatal responses to BCG vaccination are only poorly understood. ARS scientists at Beltsville, Maryland, therefore worked with researchers at Colorado State University to determine that piglets and human infants respond similarly to BCG vaccination. By affirming that the pig serves as an effective neonatal animal model for TB vaccine development, this work advanced progress towards better human vaccination to a major global disease.
2. Expanded tools to measure swine immunity. Too few tools are available to analyze disease and vaccine responses in pigs. ARS scientists at Beltsville, Maryland, therefore worked with commercial partners to clone and express immune proteins using the yeast expression system and to develop monoclonal antibodies (mAbs) to these proteins. These mAbs were characterized in collaboration with researchers at the Ohio State and Tennessee State Universities and the University of Bristol, U.K. Panels of mAbs reactive with 9 different swine immune proteins (cytokines and chemokines) were characterized; confirmed sets of mAbs are being tested for commercialization. These mAbs will enable each protein to be quantified in body fluids and enable their intracellular expression to be measured. Tools and reagents generated by this project support swine immune, disease, and biomedical research efforts worldwide.
Review Publications
Kyu-Sang, L., Dong, Q., Moll, P., Vitkovska, J., Wiktorin, G., Bannister, S., Daujotyte, D., Tuggle, C.K., Lunney, J.K., Plastow, G., Dekkers, J. 2019. BMC Genomics. 20:741. https://doi.org/10.1186/s12864-019-6122-2.
Ramos, L., Obregon-Henao, A., Henao-Tamayo, M., Bowen, R., Izzo, A., Lunney, J.K., Gonzalez-Juarrero, M. 2019. Minipigs as neonatal animal model for tuberculosis vaccine efficacy testing. Veterinary Immunology and Immunopathology. 215:109884. https://doi.org/10.1016/j.vetimm.2019.109884.
Benfield, D., Lunney, J.K., Murtaugh, M., Nelson, E., Osorio, F., Pogranichniy, R., Ramamoorthy, S., Rowland, R.R., Zimmerman, J.J., Zuckermann, F. 2020. The NC229 multi-station research consortium on emerging viral diseases of swine: solving stakeholder problems through innovative science and research. Virus Research. 280:197898. https://doi.org/10.1016/j.virusres.2020.197898.
Dawson, H.D., Sang, Y., Lunney, J.K. 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.
Hammer, S.E., Ho, C., Ando, A., Rogel-Gaillard, C., Charles, M., Tector, M., Joseph, T.A., Lunney, J.K. 2020. Importance of the MHC (SLA) in swine health and biomedical research. Review Article. 8:171-198. https://doi.org/10.1146/annurev-animal-020518-115014.
Chase, C., Lunney, J.K. 2019. Immune System. In:Zimmerman J, Karriker L, Ramirez A, Schwartz K, Stevenson G, Zhang J., editors. Diseases of Swine. 10th edition. Hoboken, New Jersey: John Wiley and Sons, Inc. p. 264-290.
Koltes, J.E., Cole, J.B., Clemmens, R., Dilger, R.N., Kramer, L.M., Lunney, J.K., Mccue, M.E., Mckay, S., Mateescu, R., Murdoch, B.M., Reuter, R., Rexroad III, C.E., Rosa, G.J.M., Serao, N.V.L., White, S.N., Woodward Greene, M.J., Worku, M., Zhang, H., Reecy, J.M., editors. 2019. A vision for development and utilization of high-throughput phenotyping and big data analytics in livestock. Frontiers in Genetics. 10:1197. https://doi.org/10.3389/fgene.2019.01197.
Entrican, G., Lunney, J.K., Wattegedera, S.R., Mwangi, W., Hope, J.C., Hammond, J.A. 2020. The veterinary immunological toolbox: past, present and future. Frontiers in Immunology. 11(1651). https://doi.org/10.3389/fimmu.2020.01651.
