Progress 02/01/15 to 01/31/19
Outputs
Target Audience:Immune reagent development is an essential tool generation step for improving research in infectious and non-infectious diseases of the horse. The immune tools generated during this project are needed to advance vaccine development, management and treatment practices of equine diseases. They are also important to provide a better understanding of immune responses of healthy horses and those that are susceptible to or protected from diseases. Althoughimmune reagents are primarily basic research tools and possiblyin a very few instances therapeutic agents, the use of an improved panel of immune reagents in basic and clinical researchcanin the midto long-termbroadlyimprove equine health and effectively support the equine industry. The immediate target audiences for equine immune reagent development are (1) the equine research community including basic and clinical researchers, (2) entities interested in commercializing immune reagents or assay based on the new tools, and (3) the wider biomedical research field interested in comparative studies, largeanimal models for human diseases, or One Health approaches. During this project theequine research community including basic and clinical researcherswas reached by (i)directly providing immune reagents from Cornell University,(ii) by offering immune reagent-based assays to determine specific immune parameters for immunological research studies through the Animal Health Diagnostic Center (AHDC) at Cornell, and (iii) by engaging incollaborative research projects and studies that included the use of the immune reagents with researchers in the US and internationally. Detailed information in respect to (i) is provided under 'Other Products'. Regarding (ii), commercial companies have not yet been seriously interested in horse reagents. This is likely due to the small market for equine immune reagents. We have not gotten any request from companies during this reporting period. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Susanna Babasyan, Post-doctoral associate, worked on the project full-time during the past year. Susanna has previously performed the recombinant protein expression and purification steps during US-VIRN. For this project, she continued with these tasks. She also learned and became very proficient in mouse immunization and the hybridoma development and characterization process. Christiane Schnabel another Post-doctoral associate in Dr. Wagner's group, also contributed to the hybridoma development and has spent part of her time to develop and characterize the new IgA, CCL3, CCL5, CXCL10, and TNF-a mAbs. Christiane published 3 peer-reviewed articles on the development of these mAbs. She was supported by other resources. Ellie Larson, PhD student in Dr. Wagner's group, has worked on the IL-13 and IL-8 mAbs. Ellie was interested in learning the methods of making and characterizing mAbs. She successfully made the new IL-8 mAb and is working on a publication for this antibody. Ellie was supported by other resources. Svenja Maier, a Cornell Leadership student from Hannover, Germany, spent ten weeks of summer research in my group in 2016. Svenja worked together with Christiane on the identification of the preferred CCL3 mAb pair and the initial steps of using them in a fluorescent bead-based assay. Svenja learnedvarious methods and techniques for mAb characterization and multiplex assay development. She was funded through the Cornell Leadership Program. Franziska Kaiser, a Cornell Leadership student from Hannover, Germany, and Steven Young, an incoming Cornell DVM student and Veterinary Investigator participant at Cornell, spent ten weeks of summer research in my group in 2015. Both, students learnedvarious methods and techniques for mAb characterization and how to prove their specificity to their respective target. They were funded through their summer research programs. How have the results been disseminated to communities of interest?New reagents developed during this project were first characterized at Cornell for specificity and recognition of the native target protein. Many of the mAbs were also sent out for testing at a second location (expert labs). Typically additional methods and applications were tested in these expert labs (e.g. immunohistochemistry). If successful, the results will be published collaboratively in peer-reviewed journals. All mAbs became available through Cornell University to the entire equine research community. For many of them articles on the primary reagent development and characterization were published. Almost all new mAbs, with the exception of those in the final characterization step, were added to our reagent website (http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm). This website lists the reagents that are currently available to all researcher through an MTA process. We established this process previously and used it for the available reagents that are listed on this website. Some of the reagents were also be used for assay development at Cornell. For example, the equine 5-plex cytokine assay mentioned above is a spin-off of the US-VIRN reagent production (https://ahdc.vet.cornell.edu/docs/Equine_Cytokine_5plex_assay.pdf). This assay is available through the AHDC at Cornell (https://ahdc.vet.cornell.edu/sects/Serol/). In addition, the equine sCD14 assay is available at the AHDC since 2016. The ADHC is an AAVLD accredited diagnostic laboratory and performs fee-for-service testing. Assays developed with the new reagents from this project are available to all equine researchers, nationally and internationally, through the AHDC. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This NIFA Equine Immune Reagent Development project (2015-2019) has addressed a total of 14 monoclonal antibody (mAb) targets in a total of 20 immunizations and cell fusions. These targets are (NCR2, CD19, CD3, IgA, CCL3, CCL5, CXCL9, CXCL10, IFN-b, IL-1b, IL-2, IL-8, IL-13, and TNF-a). Several of the targets resulted in specific mAbs and new multiplex assays, others were not successful. 'No specific mAb' is a possible outcome of mAb development and is as time consuming as the successful process. Repeats of the immunization and fusion process was performed for six of these targets and resulted for some of them in successful mAb development. During this project, new mAbs were developed at Cornell for IgA, CCL3, CCL5, CXCL10, IL-2, TNF-a, IL-1b, and IL-8. Most of these resulted in several mAbs and mAb pairs for secreted proteins. For the latter, multiplex assays for sCD14, chemokines (CCL2, CCL3, CCL5, CCL11), IL-2, TNF-a and IL-1b were established and validated. The characterization of NCR2, CD19 and IL-8 mAbs are still ongoing. A total of 24 authored or co-authored peer-reviewed publications related to equine immune reagents were published between 2015 and 2018. These publications include primary reagent development reports and articles in which the mAbs majorly contributed to the scientific outcome of the research project [1-16 add]. A total of 114 mAbs were sent to 20 different equine research groups mostly in the US but also in Switzerland, Iceland, Germany, Australia, Ireland, and Canada. These were purified and or conjugated, well characterized mAbs with proven specificity for the native equine target molecule. Equine cytokine mAbs developed at Cornell have been used to develop fluorescent bead-based multiplex assays for detection of soluble cytokines in samples from horses or cell culture supernatants. The validated assays are available to the equine research community through the Animal Health Diagnostic Center (AHDC) at Cornell University. The assays are available for a discounted research fee covering the supply and labor costs of the assay. The 5-plex cytokine multiplex assay for equine IL-4, IL-10, IL-17, IFN-a, and IFN-g, has been validated prior to this project. Around 5600 samples from different equine research groups around the world have been submitted to Cornell to be analyzed with this assay between 2015-2017. An IL-2 assay has also been developed. The assay for sCD14 has been validated and became available in 2016 during this project. More than 500 samples were measured for sCD14 at the AHDC. A multiplex assay for equine chemokines (CCL2, CCL3, CCL5, CCL11, TNF-a and IL-1b) has been established and in its final validation process. This assay has a very high potential for advancing equine immunology by improving our understanding of inflammatory responses of the horse. We consider it a real highlight of the reagent development during the ongoing project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Schnabel CL, Wemette M, Babasyan S, Freer H, Baldwin C, Wagner B. 2018. C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies. Vet. Immunol. Immunopathol., 204: 28-39.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR. 2018. Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood. Vet Immunol Immunopathol, 199: 22-31.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Schnabel CL, Wimer CL, Perkins G, Babasyan S, Freer H, Watts C, Rollins A, Osterrieder N, Wagner B. 2018. Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity. BMC Vet Res, 14: 245.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bechert U, Rohde J, Freer H, Wagner B. 2018. IgG4/7 responses correlate with contraception in mares vaccinated with SpayVac. Theriogenology, 121: 168-174.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Schnabel CL, Babasyan S, Freer H, Wagner B. 2019. CXCL10 production in equine monocytes is stimulated by interferon-gamma. Vet Immunol. Immunpathol. 207: 25-30
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wimer CL, Schnabel CL, Perkins G, Babasyan S, Freer H, Stout AE, Rollins A, Osterrieder N, Goodman LB, Glaser A, Wagner B. 2018. The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses. Plos One, 13(11):e0206679.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Perkins G, Babasyan S, Stout AE, Freer H, Rollins A, Wimer CL, Wagner B. 2019. Intranasal IgG4/7 antibody responses protect horses against equid herpesvirus-1 (EHV-1) infection including nasal virus shedding and cell-associated viremia. Virology, 531: 219-232
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Ziegler A, Hamza E, Jonsdottir S, Rhyner C, Wagner B, Sch�pbach G, Svansson V, Torsteinsdottir S , Marti E. 2018. Longitudinal analysis of allergen-specific IgE and IgG subclasses as potential predictors of insect bite hypersensitivity following first exposure to Culicoides. Vet Dermatol, 29:51-e22.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Jonsdottir S, Stefansdottir SB, Kristinarson SB, Svansson V, Bjornsson JM, Runarsdottir A, Wagner B, Marti E, Torsteinsdottir S. 2018. Barley produced Culicoides allergens are suitable for monitoring the immune response of horses immunized with E.coli expressed allergens. Vet Immunol Immunopathol, 201: 32-37.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Witonsky S, Buechner-Maxwell V, Santonastaso A, Pleasant R, Werre S, Wagner B, Ellison S, Lindsay D. 2019. Can levamisole upregulate the equine cell mediated macrophage dendritic cell T-helper 1 (CD4) T-cytotoxic (CD8) immune response in vitro? JVIM, 33: 889-896.
|
Progress 02/01/17 to 01/31/18
Outputs
Target Audience:Immune reagent development is an essential tool generation step for improving research in infectious and non-infectious diseases of the horse. The immune tools generated during this project are needed to advance vaccine development, management and treatment practices of equine diseases. They are also important to provide a better understanding of immune responses of healthy horses and those that are susceptible to or protected from diseases. Althoughimmune reagents are primarily basic research tools and possiblyin a very few instances therapeutic agents, the use of an improved panel of immune reagents in basic and clinical researchcanin the midto long-termbroadlyimprove equine health and effectively support the equine industry. The immediate target audiences for equine immune reagent development are (1) the equine research community including basic and clinical researchers, (2) entities interested in commercializing immune reagents or assay based on the new tools, and (3) the wider biomedical research field interested in comparative studies, largeanimal models for human diseases, or One Health approaches. During this reporting period theequine research community including basic and clinical researcherswas reached by (i)directly providing immune reagents from Cornell University,(ii) by offering immune reagent-based assays to determine specific immune parameters for immunological research studies through the Animal Health Diagnostic Center (AHDC) at Cornell, and (iii) by engaging incollaborative research projects and studies that included the use of the immune reagents with researchers in the US and internationally. Detailed information in respect to (i) is provided under 'Other Products'. Regarding (ii), commercial companies have not yet been seriously interested in horse reagents. This is likely due to the small market for equine immune reagents. We have not gotten any request from companies during this reporting period. Nevertheless, in 2017 a new assay for equine IgA and a multiplex assay for the equine chemokines CCL2, CCL3, CCL5 and CCL11 were established. The assays were validated and will the chemokine assay be offered through the AHDC to all equine researcher and clinicians. The CCL2 and CCL11 antibodies for this assay were previously developed during US-VIRN. Regarding (iii), two peer-reviewed publications on IgA and IL-2 mAb production, characterization and assay development were published during this reporting period. In addition, five collaborative publications are stated in the next section showing the scientific outcomes from using our former and current reagents as part of disease pathogenesis and host immune response work for horses. These mAbs were either generated as part of US-VIRN or this equine reagent development grant. Currently, another monoclonal antibody (mAb) development and characterization paper for the CCL2, CCL3, CCL5 and CCL11 mAbs combined is close to the submission stage.The production, quality testing and distribution of these immune reagent tools were continued during the third year of this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Susanna Babasyan, Post-doctoral associate, worked on the project full-time during the past year. Susanna has previously performed the recombinant protein expression and purification steps during US-VIRN. For this project, she continued with these tasks. She also learned and became very proficient in mouse immunization and the hybridoma development and characterization process. Christiane Schnabel another Post-doctoral associate in my group, also contributed to the hybridoma development and has spent part of her time to develop and characterize the new CCL3, CCL5, and CXCL10 mAbs. Christiane was supported by other resources. How have the results been disseminated to communities of interest?New reagents developed during this project will first be characterized at Cornell for specificity and recognition of the native target protein. Many of the mAbs have also been sent out for testing at a second location (expert labs). Typically additional methods and applications are tested in these expert labs. If successful, the results will be published collaboratively in peer-reviewed journals. All mAbs become available through Cornell University to the entire equine research community as soon as the manuscript is accepted and the publication is available online. At the same time the new mAbs will be added to our reagent website (http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm). This website lists the reagents that are currently available to all researcher through an MTA process. We established this process previously and used it for the available reagents that are listed on this website. Some of the reagents will also be used for assay development at Cornell. For example, the equine 5-plex cytokine assay mentioned above is a spin-off of the US-VIRN reagent production (https://ahdc.vet.cornell.edu/docs/Equine_Cytokine_5plex_assay.pdf). This assay is available through the AHDC at Cornell (https://ahdc.vet.cornell.edu/sects/Serol/). In addition, the equine sCD14 assay is available at the AHDC since 2016. The ADHC is an AAVLD accredited diagnostic laboratory and performs fee-for-service testing. Assays developed with the new reagents from this project are available to all equine researchers, nationally and internationally, through the AHDC. What do you plan to do during the next reporting period to accomplish the goals?(1) We will finish with the fusions and characterization all mAbs described above and identify expert labs to tests the regents at a second testing site. If successful, these reagents will become available to the equine research community through Cornell using the same methods as for the existing reagents (2) Make the equine chemokine assay (CCL2, CCL3, CCL5 and CCL11) available to the community in 2018. This has been delayed due to other priorities in 2017. These chemokines are very sensitive and reliable tool to detect inflammation in horses. It can be expected that chemokine assay will provide excellent biomarkers and prognosticators for many inflammatory diseases and conditions in horses. (3) Add mAbs against TNF-a to the chemokine assay and mAbs to IL-2 to the cytokine assay. If other mAb developments are successful they will also be added to respective assays. (4) Continue to publish the successful mAb developments.
Impacts
What was accomplished under these goals?
During the third year of this project, we continued to work on some immune targets that have been cloned but were not completed during US-VIRN, continued to develop mAb pairs for detection of soluble cytokines and chemokines, and worked on new targets. These reagents were on the preliminary priority list for this project. The new immune reagents we have started, continued or finished to develop in the third year of this project are listed below with a short summary oftheir current status. anti-equine CCL3:CCL3 is a chemokine involved in the innate immune response; a single CCL3 mAb resulted from a fusion using yeast expressed equine CCL3 during US-VIRN. To obtain a pair of mAbs that is suitable for a CCL3 secretion assay an IL-4/CCL3 fusion protein was expressed in mammalian cells in year 1. Immunization of a mouse and a cell fusion was performed and 10 CCL3-specific mAb clones were obtained, purified and biotinylated. In year 2, we selected the best mAb pair for a CCL3 secretion assay. The assay was established and optimized in year 3 and the CCL3 assay was added to an equine chemokine multiplex assay. This assay has been validated. It includes equine CCL2, CCL3, CCL5, and CCL11 and will be made available to the research community before the end of this project. The CCL3 mAbs have also been characterized for flow cytometry. A manuscript including the CCL3 mAb production and characterization is close to submission. anti-equine CCL5:CCL5 is another chemokine and cell-attractant during innate immune responses; a single, weak CCL5 mAb resulted from a fusion using yeast expressed equine CCL5 during US-VIRN. In year 1, an IL-4/CCL5 fusion protein was expressed in mammalian cells to obtain a pair of high affinity mAbs for a CCL5 secretion assay. In year 2, several CCL5 mAbs were obtained after immunization and cell fusion. The mAbs were tested by ELISA and flow cytometry. A pair of mAb for a CCL5 secretion assay was identified and added to the chemokine multiplex assay (see CCL3). A manuscript including the CCL5 mAb production and characterization is in preparation. Anti-equine CXCL10: Mabs against equine CXCL10 were produced with a CXCL10/IgG fusion protein for immunization. The fusion resulted in one mAb to equine CXCL10 which was characterized and can be used for flow cytometric analysis. A short communication is in preparation for this antibody. Anti-equine CXCL9: Mabs against equine CXCL9 are targeted using CXCL9/IL-4 fusion protein for immunization. The fusion did not result in any mAbs specific for equine CXCL9. Anti-equine IFN-ß: Mabs to anti-viral type I interferon IFN-ß were targeted using IFN-ß/IgG1 for immunization. Mabs to IFN-ß are valuable tools for studying virus infections in horses and especially for accessing local anti-viral effects in infected horses. The fusion did not result in any mAbs specific for equine IFN-ß. Anti-equine IL-2: An IL-2/IL-4 protein was used for immunization to generate a second IL-2 mAbs in addition to the published mAb (2017) for ELISA and/or multiplex assays. The fusion resulted in 6 equine IL-2 specific clones which are currently purified and further analyzed. Anti-equine TNF-a: A fusion with TNF-a/IL-4 resulted in 3 different equine TNF-a mAbs. These have been tested for specificity and resulted also in a pair of mAbs for detection of TNF-a by ELISA and in a Luminex assay. The assays are further optimized and tested for multiplexing with one of the existing assays. Anti-equine IL-8, IL-1b, IL-13 and CD19: Immunizations with these equine proteins were previously all performed in BALB/c mice, resulted in low titers and did not result in any specific antibodies. We decided to use the same IL-4 fusion proteins of these targets again in mice with a different genetic background to improve immune recognition. The immunization for all 4 proteins is ongoing in Black6 mice.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
The following two articles describe the original mAb development and characterization by our group:
Schnabel CL, Babasyan S, Freer H, Wagner B. 2017. Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay. Vet Immunol Immunopathol, 188: 12-20.
Freer H, Hillegas JM, Wimer C, Baldwin C, LaBresh J, Wagner B. 2017. A monoclonal antibody for detection of intracellular and secreted interleukin-2 in horses. Vet Immunol Immunopathol, 191: 30-35.
The next articles have used equine reagents produced by our group and acknowledge NIFA support for these reagents in the article:
Wagner B, Perkins G, Babasyan S, Freer H, Keggan A, Gooman LB, Glaser A, Torsteinsd�ttir S, Svansson V, Bj�rnsd�ttir S. 2017. Neonatal immunization with a single IL-4/antigen dose induces increased antibody responses after challenge infection with equine herpesvirus type 1 (EHV-1) at weanling age. PLOS ONE, 12(1):e0169072.
Pfaender S, Walter S, Grabski E, Todt D, Bruening J, Romero-Brey I, Gather T, Brown RJ, Hahn K, Puff C, Pfankuche VM, Hansmann F, Postel A, Becher P, Thiel V, Kalinke U, Wagner B, Bartenschlager R, Baumg�rtner W, Feige K, Pietschmann T, Cavalleri JM, Steinmann E. 2017. Immune protection against reinfection with nonprimate hepacivirus. Proc Natl Acad Sci U S A, 114: E2430-E2439.
Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR. Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood. Vet Immunol Immunopathol, in press.
These publications used reagents developed by this project but did not acknowledge the funding source:
Carossino M, Wagner B, Loynachan A, Cook F, Canisso I, Chelvarajan L, Edwards C, Nam B, Timoney J, Timoney P, Balasuriya U. Equine arteritis virus elicits a mucosal antibody response in the reproductive tract of the persistently infected stallion. Clin Vacc Immunol., 24(10), pii: e00215-17.
Ziegler A, Hamza E, Jonsdottir S, Rhyner C, Wagner B, Sch�pbach G, Svansson V, Torsteinsdottir S , Marti E. 2018. Longitudinal analysis of allergen-specific IgE and IgG subclasses as potential predictors of insect bite hypersensitivity following first exposure to Culicoides. Vet Dermatol, 29:51-e22.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Oral Presentations:
Wagner B. - invited speaker. Advances in and applications of equine immunological reagents and assays. Meeting on: Outcomes of the USDA-NIFA Grant on Identification of Genetic Factors responsible for the Establishment of EAV Carrier Sate in Stallions. Marriot Griffin Gate, Gluck Equine Research Center, University of Kentucky, Lexington, KY, November 10th, 2017.
Schnabel CL, Babasyan S, Freer H, Wagner B: Quantification of equine immunoglobulin A in serum and mucosal secretions by a fluorescent bead-based assay, oral presentation, CRWAD 2017.
Posters:
Schnabel CL, Babasyan S, Freer H, Wemette M, Wagner B: Identification of C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells by newly generated monoclonal antibodies, poster, CRWAD, Chicago, IL, USA, 2017.
Schnabel CL, Babasyan S, Freer H Wemette M, Wagner B: Identification of C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells by newly generated monoclonal antibodies, poster, Annual Harry M. Zweig Memorial Fund for Equine Research Poster Session, Cornell University, NY, USA, 2017.
|
Progress 02/01/16 to 01/31/17
Outputs
Target Audience:Immune reagent development is an essential tool generation step for improving research in infectious and non-infectious diseases of the horse. The immune tools generated during this project are needed to advance vaccine development, management and treatment practices of equine diseases. They are also important to provide a better understanding of immune responses of healthy horses and those that are susceptible to or protected from diseases. Althoughimmune reagents are primarily basic research tools and possiblyin a very few instances therapeutic agents, the use of an improved panel of immune reagents in basic and clinical researchcanin the midto long-termbroadlyimprove equine health and effectively support the equine industry. The immediate target audiences for equine immune reagent development are (1) the equine research community including basic and clinical researchers, (2) entities interested in commercializing immune reagents or assay based on the new tools, and (3) the wider biomedical research field interested in comparative studies, largeanimal models for human diseases, or One Health approaches. During this reporting period theequine research community including basic and clinical researcherswas reached by (i)directly providing immune reagents from Cornell University,(ii) by offering immune reagent-based assays to determine specific immune parameters for immunological research studies through the Animal Health Diagnostic Center (AHDC) at Cornell, and (iii) by engaging incollaborative research projects and studies that included the use of the immune reagents with researchers in the US and internationally. Detailed information in respect to (i) is provided under 'Other Products'. Regarding (ii), commercial companies have not yet been seriously interested in horse reagents. This is likey due to the small market for equine immune reagents. We have gotten two requests from companies in this reporting period. One didn't come through (company dropped out) and the second just occurred last week and is currently negotiated. Nevertheless, in 2016 a new assay for soluble CD14 (sCD14) detection in horses was validated and is now offered through the AHDC to all equine researcher and clinicians. The CD14 antibodies have been developed during US-VIRN. The sCD14 assay is an additional outcome using these reagents, has been validated during this project, and was used for several collaborations. For (iii), five publications are stated in the next section showing the scientific outcomes from our former reagent development work for horses as part of US-VIRN and this equine reagent development grant. In addition, one monoclonal antibody (mAb) development and characterization paper has been accepted and is in press (anti-equine IgA mAbs) and one is submitted and under review (IL-2 mAb).The production, quality testing and distribution of these immune reagent tools were continued during the second year of this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Susanna Babasyan, Post-doctoral associate, devoted 83% effort during the past year to the project.Susanna has previously performed the recombinant protein expression and purification steps during US-VIRN. For this project, she continued with these tasks. She also learned and became very proficient in mouse immunization and the hybridoma development and characterization process. Christiane Schnabel another Post-doctoral associate in my group, was also trained on the hybridoma development methods and has spent part of her time to develop and characterize the new CCL3, CCL5, and IgA mAbs. Christiane was supported by other resources. Svenja Maier, a Cornell Leadership student from Hannover, Germany, spent ten weeks of summer research in my group in 2016. Svenja worked together with Christiane on the identification of the preferred CCL3 mAb pair and the initial steps of using them in a fluorescent bead based assay. Svenja learnedvarious methods and techniques for mAb characterization and multiplex assay development. She was funded through the Cornell Leadership Program. How have the results been disseminated to communities of interest?New reagents developed during this project will first be characterized at Cornell for specificity and recognition of the native target protein. Many of the mAbs have also been sent out for testing at a second location (expert labs). Typically additional methods and applications are tested in these expert labs. If successful, the results will be published collaboratively in peer-reviewed journals. All mAbs become available through Cornell University to the entire equine research community as soon as the manuscript is accepted and the publication is available online. At the same time the new mAbs will be added to our reagent website (http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm). This website lists the reagents that are currently available to all researcher through an MTA process. We established this process previously and used it for the available reagents that are listed on this website. Some of the reagents will also be used for assay development at Cornell. For example, the equine 5-plex cytokine assay mentioned above is a spin-off of the US-VIRN reagent production (https://ahdc.vet.cornell.edu/docs/Equine_Cytokine_5plex_assay.pdf). This assay is available through the AHDC at Cornell (https://ahdc.vet.cornell.edu/sects/Serol/). In addition, the equine sCD14 assay is available at the AHDC since 2016. The ADHC is an AAVLD accredited diagnostic laboratory and performs fee-for-service testing. Assays developed with the new reagents from this project are available to all equine researchers, nationally and internationally, through the AHDC. What do you plan to do during the next reporting period to accomplish the goals?(1) We will continue with the characterization of the new mAbs above and identify expert labs to tests the regents at a second testing site. If successful, these reagents will become available to the equine research community through Cornell using the same methods as for the existing reagents (2) Make the equine chemokine assay (CCL2, CCL3, CCL5 and CCL11) available to the community in 2017. These chemokines are very sensitive and reliable tool to detect inflammation in horses. It can be expected that chemokine assay will provide excellent biomarkers and prognosticators for many inflammatory diseases and conditions in horses. (3) Add mAbs against CXCL9 and CXCL10 (if successful) to the chemokine assay and mAbs to IFN-ß to the cytokine assay.
Impacts
What was accomplished under these goals?
During the second year of this project, we still continued to work on some immune targets that have been cloned but were not completed during US-VIRN and we have worked on several new targets. These reagents were on the preliminary priority list for this project. The new immune reagents we have started, continued or finished to develop in the second year of this project are listed below with a short summary oftheir current status. The goal for year 1 was to develop 8 new mAbs. anti-equine NCR2:NCR2 is one of the NK-cell receptors; due to the lack of NK-cell markers the analysis of this innate immune cell population has been compromised; the equine NCR2 was expressed as an IL-4 fusion protein in mammalian cells; immunization of a mouse and a cell fusion was performed in year 1 of this project. In year 2,10 NCR2 mAb clones have been purified and were further characterized; preliminary results show that several of these mAbs are equine NCR2 specific when tested on the recombinant NCR2 expressed in mammalian cells. A few mAbs also detected, as expected, a low percentage of lymphoid cells in the peripheral blood. They also detect an increased number of cells with large lymphocyte morphology after IL-2 and IL-15 stimulation and an increased NCR2 positive intranasal population after infection with EHV-1. We are still doing a few additional experiments to complete this target. The publication of the development and characterization of the NCR2 antibodies is pending. anti-equine CCL3:CCL3 is a chemokine involved in the innate immune response; a single CCL3 mAb resulted from a fusion using yeast expressed equine CCL3 during US-VIRN. To obtain a pair of mAbs that is suitable for a CCL3 secretion assay an IL-4/CCL3 fusion protein was expressed in mammalian cells in year 1. Immunization of a mouse and a cell fusion was performed and 10 CCL3-specific mAb clones were obtained, purified and biotinylated. In year 2, we selected the best mAb pair for a CCL3 secretion assay. The assay was established and optimized. The CCL3 assay was added to an equine chemokine multiplex assay. This assay has been validated. It includes equine CCL2, CCL3, CCL5, and CCL11 and will be made available to the research community before the end of this project. The CCL3 mAbs have also been characterized for flow cytometry. A manuscript including the CCL3 mAb production and characterization is in preparation. anti-equine IgA:IgA mAbs are important tools for the analysis of mucosal immunity. Thus far, only one IgA mAb was available. We have used affinity-purified equine IgA to immunize a mouse and performed a fusion in year 1. Five IgA-specific mAbs were identified by ELISA and Western blotting. In year 2, the best mAb pair for detecting IgA and quantifying it in equine serum and various secretions was identified. The mAb and IgA assay development publication is in press in Vet. Immunol. & Immunopathol. anti-equine CD3:CD3 has been identified by equine researcher in the US and internationally as a high priority marker for horses; it is the marker of choice to stain all T-cells; for horses, a very good CD3 marker was developed about 20 years ago but is not readily available anymore; we have thus performed a fusion to obtain CD3 mAbs and have obtained four promising clones recognizing the CD4 and CD8 positive lymphocytes in the peripheral blood but no other lymphoid cells. The characterization of these mAbs is ongoing. anti-equine CCL5:CCL5 is another chemokine and cell-attractant during innate immune responses; a single, weak CCL5 mAb resulted from a fusion using yeast expressed equine CCL5 during US-VIRN. In year 1, an IL-4/CCL5 fusion protein was expressed in mammalian cells to obtain a pair of high affinity mAbs for a CCL5 secretion assay. In year 2, several CCL5 mAbs were obtained after immunization and cell fusion. The mAbs were tested by ELISA and flow cytometry. A pair of mAb for a CCL5 secretion assay was identified and added to the chemokine multiplex assay (see CCL3). A manuscript including the CCL3 mAb production and characterization is in preparation. Anti-equine CXCL10: Mabs against equine CXCL10 are currently in preparation. In year 2, a CXCL10/IgG fusion protein was cloned and expressed. The protein has been purified and the immunization of a mouse is currently ongoing. Anti-equine CXCL9: Mabs against equine CXCL9 are also in preparation. In year 2, a CXCL9/IgG fusion protein was cloned and expressed. The protein has been purified and the immunization of a mouse is ongoing. Anti-equine IFN-ß: The anti-viral type I interferon IFN-ß is of interest for viral infection of the horse. Mabs to IFN-ß are valuable tools for studying virus infections in horses and especially for accessing local anti-viral effects in infected horses. An IFN-ß/IgG fusion protein has been cloned and expressed. It is currently used for mouse immunization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Wagner B, Perkins G, Babasyan S, Freer H, Keggan A, Gooman LB, Glaser A, Torsteinsd�ttir S, Svansson V, Bj�rnsd�ttir S. 2017. Neonatal immunization with a single IL-4/antigen dose induces increased antibody responses after challenge infection with equine herpesvirus type 1 (EHV-1) at weanling age. PLOS ONE, 12(1):e0169072.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Pfaender S, Walter S, Grabski E, Todt D, Bruening J, Romero-Brey I, Gather T, Brown RJ, Hahn K, Puff C, Pfankuche VM, Hansmann F, Postel A, Becher P, Thiel V, Kalinke U, Wagner B, Bartenschlager R, Baumg�rtner W, Feige K, Pietschmann T, Cavalleri JM, Steinmann E. 2017. Immune protection against reinfection with nonprimate hepacivirus. Proc Natl Acad Sci U S A, 114: E2430-E2439.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2017
Citation:
Schnabel CL, Babasyan S, Freer H, Wagner B. Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay. Vet Immunol Immunopathol, in press.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Freer H, Hillegas JM, Wimer C, Baldwin C, LaBresh J, Wagner B. A monoclonal antibody for detection of intracellular and secreted interleukin-2 in horses. submitted
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Pennington MR, Curtis TM, Divers TJ, Wagner B, Ness SA, Tennant BC, Van de Walle GR. 2016. Equine mesenchymal stromal cells (MSC) from different sources efficiently differentiate into hepatocyte like cells (HLC). Tissue Eng. Part C Methods, 22: 596-607.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Fogle J, Jacob M, Blikslager A, Edwards A, Wagner B, Dean K, Fogle C. 2017. Comparison of LPS and soluble CD14 measurement between clinically endotoxaemic and nonendotoxaemic horses. Equine Vet J, 49: 155-159.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Bonelli F, Meucci V, Divers TJ, Wagner B, Intorre L, Sgorbini M. 2017. Kinetics of plasma procalcitonin, soluble CD14, CCL2 and IL-10 after a sublethal infusion of lipopolysaccharide in horses. Vet Immunol Immunopathol, 184:29-35.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:Immune reagent development is an essential tool generation step for improving research in infectious and non-infectious diseases of the horse. The immune tools generated during this project are needed to advance vaccine development, management and treatment practices of equine diseases. They are also important to provide a better understanding of immune responses of healthy horses and those that are susceptible to or protected from diseases. Althoughimmune reagents are primarily basic research tools and possiblyin a very few instances therapeutic agents, the use of an improved panel of immune reagents in basic and clinical researchcanin the midto long-termbroadlyimprove equine health and effectively support the equine industry. The immediate target audiences for equine immune reagent development are (1) the equine research community including basic and clinical researchers, (2) entities interested in commercializing immune reagents or assay based on the new tools, and (3) the wider biomedical research field interested in comparative studies, largeanimal models for human diseases, or One Health approaches. During this reporting period theequine research community including basic and clinical researcherswas reached by (i)directly providing immune reagents from Cornell University,(ii) by offering immune reagent-based assays to determine specific immune parameters for immunological research studies through the Animal Health Diagnostic Center at Cornell, and (iii) by engaging incollaborative research projects and studies that included the use of the immune reagents with researchers in the US and internationally. Detailed information in respect to (i) is provided under 'Other Products'. Regarding (ii), commercial companies have not yet been seriously interested in horse reagents. This is probably due to the small market for equine immune reagents. For (iii), seven publications were selected in the next section showing the scientific outcomes from our former reagent development work for horses as part of US-VIRN.The production, quality testing and distribution of these immune reagent tools were continued during the first year of this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Susanna Babasyan, Post-doctoral associate, worked on the project full-time during the past year. Susanna has previously performed the recombinant protein expression and purification steps during US-VIRN. For this project, she continued with these tasks. She also learned and became very proficient in mouse immunization and the hybridoma development and characterization process. Christiane Schnabel another Post-doctoral associate in my group, was also trained on the hybridoma development methods and has spent part of her time to develop and characterize the new CCL3 and IgA mAbs. Christiane was supported by other resources. Franziska Kaiser, a Cornell Leadership student from Hannover, Germany, and Steven Young, an incoming Cornell DVM student and Veterinary Investigator participant at Cornell, spent ten weeks of summer research in my group in 2015. Both, students learnedvarious methods and techniques for mAb characterization and how to prove their specificity to their respective target. They were funded through their summer research programs. How have the results been disseminated to communities of interest?The new reagents will be fully characterized at Cornell and if possible tested at a second location (expert labs). The results will then be published in peer-reviewed journals. The mAbs will become available through Cornell University as soon as the manuscript is accepted and available online. At the same time the new mAbs will be added to our reagent website (http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm). This website lists the reagents that are currently available to all researcher through an MTA process. We established this process previously and used it for the available reagents that are listed on this website. Some of the reagents will also be used for assay development at Cornell. For example, the equine 5-plex cytokine assay mentioned above is a spin-off of the US-VIRN reagent production (https://ahdc.vet.cornell.edu/docs/Equine_Cytokine_5plex_assay.pdf). This assay is available through the AHDC at Cornell (https://ahdc.vet.cornell.edu/sects/Serol/). The ADHC is an AAVLD accredited diagnostic laboratory and performs fee-for-service testing. Assays developed with the new reagents from this project will also be made available to all researchers through the AHDC. What do you plan to do during the next reporting period to accomplish the goals?(1) We will continue with the characterization of the new mAbs above and identify expert labs to tests the regents at a second testing site. If successful, these reagents will become available to the equine research community through Cornell using the same methods as for the existing reagents (2) Reach out to the equine research community within the next three months to identify additional targets for the equine priority list. The establishment of the final equine priority list was originally planned earlier (during the first year) but was delayed because of the ongoing development of proteins and mAbs that were already in the development pipeline. Several of them were also identified on the preliminary priority list for horse reagents. Several new targets will be started in the coming year. (3) Addition of CCL3 and CCL5 assays to the current chemokine assay (CCL2 and CCL11). The goal is to make the chemokine multiplex assay available to the community in 2017. The first two chemokines have shown that this will likely become a very sensitive and reliable tool to detect inflammation in horses. It can be expected that chemokines will be excellent biomarker and prognosticators for many inflammatory diseases and conditions.
Impacts
What was accomplished under these goals?
During the first year of this project, we have continued to work on new immune targets that have been cloned but were not further started during US-VIRN. Several of the year 1 targets were on the preliminary priority list for this project. The new immune reagents we have developed in the past year are listed below with a short summary oftheir current status. The goal for year 1 was to develop 4 new mAbs. anti-equine NCR2:NCR2 is one of the NK-cell receptors; due to the lack of NK-cell markers the analysis of this innate immune cell population has been compromised; the equine NCR2 was expressed as an IL-4 fusion protein in mammalian cells; immunization of a mouse and a cell fusion was performed; 10 NCR2 mAb clones have been purified and are currently further characterized; preliminary results show that several of these mAbs are equine NCR2 specific; they detect, as expected, a low percentage of lymphoid cells in the peripheral blood; they also detect an increased number of cells with large lymphocyte morphology after IL-2 and IL-15 stimulation. anti-equine CD19: CD19 is part of the B-cell co-receptor molecules and a classical B-cell marker; B-cell markers in good quality are still unavailable for horses; the equine CD19 was expressed as an IL-4 fusion protein in mammalian cells; immunization of a mouse and a cell fusion was performed; initially a single positive clone was identified that detected the recombinant CD19 and also equine B-cells; unfortunately the clone was not stable; limiting dilution cloning from an early back-up aliquot will be performed and eventually a new fusion. anti-equine CCL3: CCL3 is a chemokine involved in the innate immune response; a single CCL3 mAb resulted from a fusion using yeast expressed equine CCL3 during US-VIRN; to obtain a pair of mAbs that is suitable for a CCL3 secretion assay an IL-4/CCL3 fusion protein was expressed in mammalian cells; immunization of a mouse and a cell fusion was performed; 10 CCL3-specific mAb clones were obtained, purified and biotinylated; these are currently tested for the best mAb pair for a CCL3 secretion assay; once established and optimized, the CCL3 assay will be added to an equine chemokine multiplex assay that is currently under development and includes equine CCL2 and CCL11 assays. anti-equine IgA: IgA mAbs are important tools for the analysis of mucosal immunity; thus far only one IgA mAb was available; this did not allow to measure total IgA values in horses; we have used affinity purified equine IgA to immunize a mouse and performed a fusion; 5 IgA-specific mAbs were identified by ELISA and Western blotting; they are currently expanded for purification and further characterized for their use in total IgA and antigen-specific IgA assays. anti-equine CD3: CD3 has been identified by equine researcher in the US and internationally as a high priority marker for horses; it is the marker of choice to stain all T-cells; for horses, a very good CD3 marker was developed about 20 years ago but is not readily available anymore; we have thus performed a fusion to obtain CD3 mAbs and have obtained four very promising clones recognizing the CD4 and CD8 positive lymphocytes in the peripheral blood but no other lymphoid cells; the four mAbs are currently further characterized. anti-equine CCL5: CCL5 is another chemokine and cell-attractant during innate immune responses; a single, weak CCL5 mAb resulted from a fusion using yeast expressed equine CCL5 during US-VIRN; to obtain a pair of high affinity mAbs for a CCL5 secretion assay an IL-4/CCL5 fusion protein was expressed in mammalian cells; immunization with the purified protein is currently ongoing; the goal is to add the CCL5 assay to the equine chemokine multiplex assay (see CCL3).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Perkins GA, Wagner B. 2015. The development of equine immunity: Current knowledge on immunology in the young horse. Equine Vet. J., 47: 267-274. (VIRN)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ortved KF, Wagner B, Calcedo R, Wilson JM, Schaefer DM, Nixon AJ. 2015. Humoral and cell-mediated immune response, and growth factor synthesis after direct intra-articular injection of rAAV2-IGF-I and rAAV5-IGF-I in the equine middle carpal joint. Human Gene Therapy, 26: 161-171. (VIRN)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schnabel CL, Steinig P, Schuberth HJ, Koy M, Wagner B, Wittig B, Juhls C, Willenbrock S, Murua Escobar H, Jaehnig P, Feige K, Cavaller JMV. 2015. Influences of Age and Sex on Leukocytes of Healthy Horses and their ex vivo Cytokine Release. Vet. Immunol. Immunopathol., 165: 64-74. (VIRN)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schnabel CL, Steinig P, Koy M, Schuberth HJ, Juhls C, Oswald D, Wittig B , Willenbrock S, Murua Escobar H, Pfarrer C, Wagner B, Jaehnig P, Moritz A, Feige K, Cavalleri JMV. 2015. Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent. BMC Veterinary Research, 11:140. (VIRN)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Wagner B, Goodman LB, Babasyan S, Freer H, Torsteinsd�ttir S, Svansson V, Bj�rnsd�ttir S, Perkins GA. 2015. Antibody and cellular immune responses of na�ve mares to repeated vaccination with an inactivated equine herpesvirus vaccine. Vaccine, 33:5588-5597. (VIRN)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Korn A, Miller D, Dong L, Buckles E, Wagner B, Ainsworth DM. 2015. Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response. PLOS One, 10: e0142622. (VIRN)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Fogle J, Jacob M, Blikslager A, Edwards A, Wagner B, Dean K, Fogle C. Comparison of LPS and soluble CD14 measurement between clinically endotoxaemic and nonendotoxaemic horses. Equine Vet J, in press. (VIRN)
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