Progress 07/01/19 to 06/30/22
Outputs
Target Audience:Immune reagent development is an essential tool generation step for research in infectious and non-infectious diseases of the horse. The immune reagents 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 researchwillin 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, (iii) by engaging incollaborative research projects and studies that included the use of the immune reagents with researchers in the US and internationally, and (iv) by licensing several of the monoclonal antibody reagents developed during this project to biotechnology companies for commercial distribution. Detailed information in respect to (i) and (ii) is provided under 'Other Products'. In regards to (iv), Cornell was approached by two biotech companies interested in the commercialization of equine reagents. At this time, licensing agreements are in place in place with both companies and hybridoma clones have been provided to one company. After the initially slow progress on the commercialization process - equine immunology is a small field - we are confident that the commercialization of well validated equine immune reagents from Cornell will broaden their use in basic and clinical research and further ensure longevity of the monoclonal antibodies that were developed during this project. Changes/Problems:As an addition to the project, we have performed a successful technology transfer to apply our mAb development and characterization process that we established over many years for equine mAbs to immune reagent development in cattle. This was funded by a Federal Capacity Fund to a new faculty member and bovine researcher at our department, Dr. Sabine Mann (bovine metabolic disease researcher), and Dr. Bettina Wagner (PD of this project). We have collaborated on this project during the past three year. Dr. Anja Sipka, Research Associate, has been instrumental in performing the lab work for this approach by collaborating closely with Dr. Susanna Babasyan. Anja has successfully developed new mAbs against bovine TNF-a and a first bovine multiplex assay for TNF-a, IFN-g and IL-10. Both developments have been published in the Journal of Dairy Science Communications in 2021 (TNF-a mAbs) and 2022 (multiplex assay). The latter publication was also highlighted as 'Editor's choice' in the past month. What opportunities for training and professional development has the project provided?Susanna Babasyan, Research Associate, worked on the project full-time during the past three years. Susanna has performed the recombinant protein expression, mAb development, and protein and mAb purification steps for this project period. She is also instrumental in helping new graduate students interested in mAb production to introduce them to the methods and doing procedures with them. Susanna has also performed all shipment of mAbs in response to researcher's requests for reagents and has prepared the hybridomas for shipment to the Biotech companies after licensing agreements were signed. Ellie Larson, PhD student in Dr. Wagner's group, has worked on the IL-8 mAb development and characterization. She also made excellent use of the first IL-8 mAb she developed by identifying a new mechanistic link of IL-8 production in IgE+ monocytes which supports the ongoing inflammation and circulus virtuosos of allergic diseases. The mAb development and new finding of IL-8 synthesis in the context of allergy were published by Ellie in the prestigious Journal of Immunology. Ellie was supported by other resources. Naya Eady, another PhD graduate student in the lab, worked with Susanna on the successful IgD fusion and has taken on the characterization of the resulting IgD mAbs. She has used the IgD clones for improving the phenotyping of equine B-cells and characterization of subpopulations that represent different activation and maturation stages of the cells. Naya is preparing a manuscript about her work on IgD+ B-cells in the horse. Naya was supported by other resources. Camille Holmes, PhD graduate student, made mAbs against SLPI and granulysin. The SLPI fusion resulted in several positive clones. Camille characterized them and identified mAbs that detect the native SLPI in the horse. She also established and assay for secreted anti-inflammatory SLPI quantification. This assay is becoming an important new tool for inflammatory processes on mucosal surface of the horse. Camille also characterized the granulysin mAb as a new reagent for T cell function. Manuscripts are in preparation. Camille was supported by other resources. 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. Some of the mAbs have also been sent out for testing or use at a second location (expert labs). Typically, additional methods and applications are tested in these expert labs. If successful, the results have been published collaboratively as oral/poster presentations and in peer-reviewed journals. All characterized mAbs are 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 are added to our reagent website (https://wagnerlab.vet.cornell.edu/research/equine-reagents.html). The website lists the reagents that are currently available to all researcher through an MTA process. New mAbs are added to the reagent website once they finish the characterization steps to ensure that only high-quality reagents are distributed to equine researcher. Some of the equine reagents have 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, an equine sCD14 assay is available at the AHDC since 2016 and the 6-plex chemokine assay described in Objective 1 has been used extensively during this project for equine research. The newest assay development is the assay for equine SLPI. 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. This service is used frequently by equine researchers and clinician for a wide variety of applications in equine health and diseases. In addition, Cornell has licensed various equine mAb clones from the reagent list above to two biotechnology companies for commercialization and world-wide distribution of the equine mAbs. The licensing agreements are finalized. Hybridoma clones were sent to one company and will be sent to the second entity soon. Commercial interest for equine mAbs (smaller market) has been virtually zero before this project period. We consider it a great success of our mAb development and characterization that this has changed to reach broader distribution and allow for easy access to equine mAbs developed during USDA/NIFA projects including this one.? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 - During of this project, we have finished assay development and validation for the 6-plex chemokine assay. The assay has been used (beta-tested) during the past two year for different research collaborations including those joint diseases, equine breakdown injuries, septicemia, equine asthma, EPM, EHV-1, equine hepatitis, wound healing, and endometritis. This list shows the broad applicability that this test will have for inflammatory and infectious diseases of the horse. The current publication outcomes using this assays are listed under 'products' and the assay is described in more detail under 'other products'. Objective 2 - was the development of ten mAbs to cell surface molecules (for an improved phenotypic characterization of major equine immune cell populations. We have developed an additional B-cell marker for IgD as part of the B-cell receptor, and mAbs for equine SLPI and granulysin as markers of different T-cell activation, development and effector stages. MAbs for IL-6 and IL-8 that advance the differentiation of monocytes populations and functions are another outcome under this objective. In addition, we have cloned five additional T-cell markers for mAb production into expression vectors. The numeric outcome of mAbs under this aim is a little less than the targeted number which is typical for cell surface mAbs. Not all attempts to make a mAb for a specific target are successful. Objective 3 - was to further simplify the distribution of the mAbs and develop a sustainable access system for the equine immune reagents produced under the NIFA umbrella. During the project we provided more than 120 equine mAbs through the established and simplified Cornell MTA process to equine researchers in the US and various other countries. We will continue to distribute these high quality mAbs and provide validated immune marker assays to the entire equine research community beyond the end date of this project. In addition, Cornell has licensed several equine mAbs that were produced during the USDA/NIFA equine reagent development project to two different biotechnology companies for commercialization. We expect that the first equine mAbs will be available and widely distributed by these companies in the near future.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Simonin EM, Babasyan S, Wagner B. Peripheral CD23hi/IgE+ plasmablasts secrete IgE and correlate with allergic disease severity. J Immunol, in press
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Holmes CM, Schnabel C, Babasyan S, Eady N, Wagner B. Characterization of mucosal T cell populations in equine herpesvirus type 1 (EHV-1) infection. Poster at the 23rd Annual Upstate New York Immunology Conference. Cooperstown, NY October 18-21, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Eady N, Babasyan S, Wagner B. IgD monoclonal antibody development for phenotyping mature naïve B-cell populations in horses. Poster at the 23rd Annual Upstate New York Immunology Conference. Cooperstown, NY October 18-21, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Wagner B, Babasyan S, Larson E, Eady N, Holmes C. Monoclonal antibody development for horses: an update. Oral onsite and online presentation during CRWAD conference, Chicago, IL, December 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Larson E, Babasyan S, Wagner B. Peripheral IgE+ plasmablasts secrete IgE and correlate to allergic disease severity. Oral onsite and online presentation during CRWAD conference, Chicago, IL, December 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Larson E, Babasyan S, Wagner B. IgE-binding monocytes promote allergic inflammation through IL-8 production. Oral onsite and online presentation during CRWAD conference, Chicago, IL, December 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Eady N, Babasyan S, Wagner B. IgD monoclonal antibody development for phenotyping maturing B-cell populations in horses. Oral onsite and online presentation during CRWAD conference, Chicago, IL, December 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Babasyan S, Freer H, Wagner B. Development of anti-equine IL-1? monoclonal antibodies. Poster during CRWAD conference, Chicago, IL, December 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Holmes C, Schnabel C, Babasyan S, Eady N, Wagner B. Identification of antileukoproteinase as a marker for the immune status of horses during Equine herpesvirus type 1 infection. Poster during CRWAD conference, Chicago, IL, December 5-7, 2021.
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Immune reagent development is an essential tool generation step for 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) and (ii) is provided under 'Other Products'. In 2019, we were approached by one biotech company interested in the commercialization of some of the equine reagents. At this time, a licensing agreement is in place in place and the company has successfully tested the reagents. This was the first serious request from a biotech company for equine immune reagents. The rather low commercial interest in these tools is likely due to the small market for equine immune reagents. This may also explain the slow progress on the commercialization process. 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 performed the recombinant protein expression, mAb development, and protein and mAb purification steps during US-VIRN and our previous equine immune reagent development grant. For this project period, she continued with these tasks. She is also instrumental in helping new graduate students interested in mAb production to introduce them to the methods and doing procedures with them. Ellie Larson, PhD student in Dr. Wagner's group, has worked on the IL-8 mAb development and characterization. During the past year Ellie, did the second IL-8 fusion, which unfortunately did not work out. However, she also made excellent use of the first IL-8 mAb she developed by identifying a new mechanistic link of IL-8 production in IgE+ monocytes which supports the ongoing inflammation and circulus virtuosos of allergic diseases. The mAb development and new finding of IL-8 synthesis in the context of allergy were published by Ellie in the prestigious Journal of Immunology. Ellie is supported by other resources. Two new PhD graduate students, Naya Eady and Camille Holmes, were interested in learning the methods of making and characterizing mAbs. Naya worked with Susanna on the successful IgD fusion and is currently further characterizing the IgD mAbs. Camille made mAbs against SLPI and granulysin. Both fusions resulted in several positive clones and Camille is characterizing them further to identify those detecting native proteins and resulting in assays for secreted protein quantification. Naya and Camille are supported by other resources. How have the results been disseminated to communities of interest?New reagents developed during this project are first characterized at Cornell for specificity and recognition of the native target protein. Some of the mAbs have also been sent out for testing or use at a second location (expert labs). Typically, additional methods and applications are tested in these expert labs. If successful, the results have been published collaboratively as oral/poster presentations and in peer-reviewed journals (see products). 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 are 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, an equine sCD14 assay is available at the AHDC since 2016 and the 6-plex chemokine assay described in Objective 1 will become available soon. 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. This service is used quite a lot by equine researchers and clinician for a wide variety of applications in equine health and diseases. In addition, we received a request from a commercial reagent supplier in 2019 to obtain some of the equine mAbs. The licensing agreement was finalized, aliquots of the reagents were successfully tested by company. These mAbs will hopefully become available soon through this mechanism. This is the first time a biotech company expressed a serious interest in obtaining and selling the equine mAbs. The lack of interest is likely due to the relatively small market for equine reagents. What do you plan to do during the next reporting period to accomplish the goals?(1) For Objective 1, We will make the equine 6-plex chemokine assay (IL-1b, TNF-a, CCL2, CCL3, CCL5 and CCL11) available to the community as a tool to measure innate and inflammatory immune reactions of the horse. This was originally planned for the past year but we realized that we should have normal value ranges for all markers defined before releasing the assay. The normal ranges for all six markers in horse serum were established in the past year. These six inflammatory markers are very sensitive and reliable tool to detect inflammation in horses. Based on our current results and by testing the assay with samples from horses with various diseases, we expect that chemokine assay will provide excellent biomarkers and prognosticators for many inflammatory diseases and conditions in horses. (2) For Objective 2, we will continue to characterize the IgD, SLPI, and granulysin mAbs. We will also express CD19 and additional T-cell activation marker genes for immunization and mAb production as soon as they are purified. (3) For Objective 3, we will continue to make reagents available to the equine research community through Cornell and continue our relationship with the biotech company to commercialize the equine reagents. (4) We will continue to publish the successful mAb developments and also present updates on new and existing mAbs during conferences. We will continue to maintain the website that lists all available equine reagents from Cornell. (5) Technology transfer: We have collaborated with a new faculty member and bovine researcher at our department, Dr. Sabine Mann, and her Post Doc Dr. Anja Sipka. They were interested to adapt to our expression and mAb development project to produce some bovine immune reagents. Anja has successfully developed new mAbs against bovine TNF-a and the peer-reviewed publication describing the mAb development has been accepted for publication.
Impacts
What was accomplished under these goals?
Objective 1 - During the first year of this project, we have finished the characterization and assay development for the 6-plex chemokine assay. The assay has been used (beta-tested) during the past year for different research collaborations including those joint diseases, septicemia, EPM, wound healing, and endometritis. This list already shows the broad applicability that this test will have once officially offered. We have also used it for EHV-1 infections and it has excellent performance for this purpose as well. During the second year of the project, the 6-plex chemokine assay has been finally validated and normal serum values ranges were developed for each chemokine to meet diagnostic accreditation criteria and then offered to the public through the Animal Health Diagnostic Center within the next year. Collaborations were ongoing in year 2 and results in several publications using this assay. The characterization of two additional mAbs to improve the analysis of cell-mediated immunity and inflammation has been continued. Two immunizations have been performed for IL-8 to obtain a second mAb to measure secreted IL-8 and for IL-6. Unfortunately, the second IL-8 fusion did not result in another mAb. IL-6 mAbs are still tested. There is at least one IL-6 mAb detecting native equine IL-6. Objective 2 - is the development of ten mAbs to cell surface molecules defined by equine researchers for an improved phenotypic characterization of major equine immune cell populations. A mAb priority list has been developed. We have developed an additional B-cell marker for IgD as part of the B-cell receptor in the second year. We also developed mAbs for equine SLPI and granulysin as markers of different T-cell activation, development and effector stages. In addition, we have cloned and are expressing five additional T-cell markers for mAb production. Objective 3 - to further simplify the distribution of the mAbs and develop a sustainable access system for the equine immune reagents produced under the NIFA umbrella. We will continue to distribute high quality mAbs and provide validated immune marker assays to the entire equine research community. During the past year, 26 equine mAbs were provided through the Cornell MTA process to equine researchers in different countries. In addition, we continue to work with a large biotech company interested in commercializing some of the equine reagents. A licensing agreement is finalized to commercialize these mAbs. The company has successfully tested the respective mAbs. We hope that these mAbs will be widely distributed soon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Larson EM, Babasyan S, Wagner B. 2021. IgE-binding monocytes have an enhanced ability to produce IL-8 (CXCL8) in animals with naturally occurring allergy. J Immunol, 206: 2312-2321.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schnabel CL, Babasyan S, Freer H, Larson EM, Wagner B. 2021. New mAbs facilitate quantification of secreted equine TNF-? and flow cytometric analysis in monocytes and T cells. Vet Immunol Immunopathol, 238:110284. doi: 0.1016/j.vetimm.2021.110284. Epub 2021 Jun 10.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Tukia E, Wagner B, Vainio K, M�nki J, Kareskoski M. 2021. The effect of uterine lavage on soluble CD14 (sCD14), chemokine ligand 2 (CCL2) and interleukin (IL)-10 levels in mares with postpartum metritis. JEVS, 98: 103365.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Raza F, Babasyan S, Larson EM, Freer HF, Schnabel CL, Wagner B. 2021. Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses. PlosOne, 16(5):e0252243. doi.org/10.1371/journal.pone.0252243.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Watkins A, Fasanello D, Stefanovski D, Schurer S, Caracappa K, DAgostino A, Costello E, Freer H, Rollins A, Read C, Su J, Colville M, Paszek M, Wagner B, Reesink H. 2021. Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models. BMC Vet Res, 17:189
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Marx C, Gardner S, Harman RM, Wagner B, Van de Walle GR. 2021. Mesenchymal stromal cell secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes. STEM CELLS Translational Medicine, online.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Fasanello D, Su J, Deng S, Yin R, Colville MJ, Berenson J, Kelly C, Freer H, Rollins A, Wagner B, Rivas F, Hall AR, Paszek MJ, Reesink H. 2021. Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-?-TSG-6-mediated HC-HA formation. Arthritis Res Ther, 23(1):218. doi: 10.1186/s13075-021-02588-7
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Elizabeth M. Larson, Susanna Babasyan, Bettina Wagner. IgE-binding Monocytes have an enhanced ability to produce IL-8 (CXCL8) in equine allergy. Online presentation during 2020 virtual CRWAD conference, December 5-8, 2020.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Susanna Babasyan, Christiane L. Schnabel, Elizabeth Larson, Bettina Wagner. Development of equine immune reagents. Online presentation during 2020 virtual CRWAD conference, December 5-8, 2020.
|
Progress 07/01/19 to 06/30/20
Outputs
Target Audience:Immune reagent development is an essential tool generation step forresearch 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) and (ii) is provided under 'Other Products'. In addition, we were approached by one biotech company interested in the commercialization of some of the equine reagents. There is an agreement in place and the company is currently testing the reagents. This was the first serious request from a biotech company. The rather low commercial interest in these tools is likely due to the small market for equine immune reagents. 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 with the exception of the COVID-19 related lab-shut down in spring 2020. Susanna has performed the recombinant protein expression, mAb development, and protein and mAb purification steps during US-VIRN and our previous equine immune reagent development grant. For this project, she continued with these tasks. Ellie Larson, PhD student in Dr. Wagner's group, has worked on the IL-8 mAb development and characterization. Ellie was interested in learning the methods of making and characterizing mAbs. She successfully made the new IL-8 mAb and wrote the manuscript for this antibody. She also worked on the development of a second IL-8 mAb to obtain a pair for detection of secreted IL-8. For this second attempt, she used the first fully characterized IL-8 mAb to make and affinity purification column and then purified native IL-8 from a bronchial lavage of a horse with recurrent airway obstruction as described above. Ellie has been supported by other resources. How have the results been disseminated to communities of interest?New reagents developed during this project are first 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 are 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, an equine sCD14 assay is available at the AHDC since 2016 and the 6-plex chemokine assay described in Objective 1 will become available this coming year. 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. In addition, we received a request from a commercial reagent supplier to obtain some of the equine mAbs. The contract has been drafted, aliquots of the reagents have been sent, and the company is currently testing the mAbs. If all goes well some of the equine mAbs will become available through this mechanism. This is the first time a biotech company expressed a serious interest in obtaining and selling the equine mAbs. The lack of interest is likely due to the relatively small market for equine reagents.? What do you plan to do during the next reporting period to accomplish the goals?(1) For Objective 1, We will make the equine 6-plex chemokine assay (IL-1b, TNF-a, CCL2, CCL3, CCL5 and CCL11) available to the community as a tool to measure innate and inflammatory immune reactions of the horse. These six inflammatory markers are very sensitive and reliable tool to detect inflammation in horses. Based on our results this past year when the assay was beta-tested with samples from horses with various diseases, we expect that chemokine assay will provide excellent biomarkers and prognosticators for many inflammatory diseases and conditions in horses. If pairs of IL-8 and IL-6 mAbs are obtained from the ongoing mAb development in Objective 1, these will be added to the assay. (2) For Objective 2, we will characterize the IgD mAbs, express CD19 for immunization and mAb production, and clone the T-cell activation marker genes for protein expression. These will be used for immunization of mice and mAb production as soon as they are purified. (3) For Objective 3, we will continue to make reagents available to the equine research community through Cornell and continue our relationship with the biotech company to commercialize the equine reagents. (4) We will continue to publish the successful mAb developments and also present updates on new and existing mAbs during conferences. We will continue to maintain the website that lists all available equine reagents from Cornell.
Impacts
What was accomplished under these goals?
Objective 1 - During the first year of this project we have finished the characterization and assay development for the 6-plex chemokine assay. The assay has been used (beta-tested) during the past year for different research collaborations including those joint diseases, septicemia, EPM, wound healing, and endometritis. This list already shows the broad applicability that this test will have once officially offered. We have also used it for EHV-1 infections and it has excellent performance for this purpose as well. The 6-plex chemokine assay will be finally validated to meet diagnostic accreditation criteria and then offered to the public through the Animal Health Diagnostic Center within the next year. The generation of two additional mAbs to improve the analysis of cell-mediated immunity and inflammation has also been started. Two immunizations have been performed for IL-8 to obtain a second mAb to measure secreted IL-8 and for IL-6. IL-8: Only one mAb was obtained using IL-4/IL-8 for immunization previously. This mAb was fully characterized during this reporting period for flow cytometric use. To obtain a second mAb for an IL-8 secretion assay, we used a different immunization strategy. Mice were immunized with a mixture of IL-4/IL-8, IL-8/IgG1 and native IL-8 to present additional immunogenic epitopes to the immune system of the mouse. We made an IL-8 affinity purification column by using the first fully characterized IL-8 mAb. Native IL-8 was then purified from a bronchial lavage of a horse with recurrent airway obstruction. IL-6: An IL-4/IL-6 fusion protein was expressed and purified and used for immunization to make mAbs. Objective 2 - is the development of ten mAbs to cell surface molecules defined by equine researchers for an improved phenotypic characterization of major equine immune cell populations. A preliminary mAb priority list has been developed. We have started the development of additional pan-B-cell marker by addressing CD19 and IgD. In addition, we will clone and express different T-cell activation markers for mAb production. During the first year of the project, IgD was cloned and expressed and mAb production is currently ongoing. The initial characterization of the early mAb supernatants showed that we may have three different mAbs recognizing native IgD as part of the B-cell receptor on B-cell surfaces. The first clones were frozen. The mAbs still need to undergo the cell culture selection and cloning processes. Objective 3 - to further simplify the distribution of the mAbs and develop a sustainable access system for the equine immune reagents produced under the NIFA umbrella. We will continue to distribute high quality mAbs and provide validated immune marker assays to the entire equine research community. During the past year, 51 equine mAbs were provided through the Cornell MTA process to equine researchers in different countries. In addition, we were approached by a large biotech company interested in commercializing some of the equine reagents. During the past couple of months an agreement has been worked out to commercialize these mAbs. The company is currently testing the respective mAbs. We hope that this will all work out on their side and these mAbs will be widely distributed soon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Larson EM, Babasyan S, Wagner B. 2020. Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity. PlosOne, 15(5):e0233537. doi: 10.1371/journal.pone.0233537.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2019
Citation:
Larson EM, Babasyan S, Wagner B. IL-8 production by IgE-binding cells in allergic horses. Biomedical and Biological Science Work in Progress Series, Cornell University, 10/30/19
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