Source: WASHINGTON STATE UNIVERSITY submitted to
IMMUNE REAGENTS FOR RUMINANTS, WITH PRIMARY FOCUS ON BOVINE-SPECIFIC REAGENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1016686
Grant No.
2018-67015-28744
Project No.
WNVdav2018
Proposal No.
2016-09336
Multistate No.
(N/A)
Program Code
A1223
Project Start Date
Mar 15, 2019
Project End Date
Mar 14, 2022
Grant Year
2019
Project Director
Davis, W. C.
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Vet Micro/Path
Non Technical Summary
The Wash. State University Monoclonal Antibody Center was established in 1982 to exploit the use of monoclonal antibody (mAb) technology to advance research in the study of the mechanisms regulating the immune response to pathogens and parasites in food and companion animals. Since its initiation, the center has developed mAbs for use in cattle (126), sheep (48), Goats (57), water buffalo (60 anti-bovine mAbs cross reactive with buffalo), pigs (60), llama/alpaca (26), and horse (28). All the mAbs are currently available to the research community through the center: http://vmp.vetmed.wsu.edu/resources/monoclonal-antibody-center , Kingfisher biotech, and Novus Biological. The reagents are also available for re-sale through international distributors on request. The mAbs are also listed through the Veterinary Immune Reagent Network (US VIRN, www.vetimm.org). Many of the advances made in understanding the composition and function of immune systems in the mentioned species have involved the use of the reagents made available through the center. Our current goal is to make use of the USDA NIFA grant to continue our efforts to develop mAb reagents critically needed to elucidate the mechanisms regulating the immune response to infectious agents and parasites in cattle. The endeavor will include development of mAbs to phylogenetically conserved epitopes expressed on orthologues of leukocyte differentiation molecules, cytokines, and chemokines to increase the utility of the mAbs for use in comparative analysis of the immune response in cattle and other ruminants. The additional reagents will increase our ability to conduct in depth studies on the immune response to pathogens and vaccines.OBJECTIVES: Cattle are one of the major livestock species used as a source of food and dairy products worldwide. Water buffalo, sheep and goats are also important livestock species. Many of the disease problems affecting livestock include these species. Understanding the mechanisms regulating the immune response to the pathogens is essential for developing a rational approach to develop vaccines for the respective pathogens. We have made most of the mAbs needed to identify the lymphocyte cell subsets involved in immune responses in the mentioned species. Our overall objective now is to develop mAbs to fill in the gaps in reagents needed to fully dissect the immune responses to pathogens and validate the functional efficacy of candidate vaccines under development. MAb development will focus on cattle as the target species. 1) Where possible conserved regions of cell membrane molecules will be used to develop mAbs. Development of mAbs to conserved epitopes will help expand opportunities for research to the other important ruminant species. 2) Optimize and make available to the research community, assays we have developed to study antigen processing and presentation by antigen presenting cells in vitro. 3) Make available, in vitro assays we have developed to directly characterize the functional activity of effector T cells proliferating in response to antigens processed and presented by antigen presenting cells. This breakthrough technology affords opportunity to obtain information on the potential efficacy of vaccines under development before testing in the field.APPROACH: Targets for development of critically needed mAbs have been identified through our ongoing research and needs identified by collaborators and the research community. These include: CD83, CD95, CD152, CD154, CD178, CD192, CD195, CD196, CD197, CD273, CD274, CD279, CD223, CD282, CD284, CD366, IL-22, IL-12R, IL-23R. The focus is on making mAbs to membrane molecules involved in regulating the immune response. New England Peptides is a company currently being used to make peptide sequences corresponding to conserved sequences on bovine and other ruminant species chemokine receptors involved in immune regulation. They will be used to develop peptides to chemokine receptors listed in the table of the proposal for mAb development. Through collaborative arrangements, Kingfisher Biotech (as mentioned in the proposal) will provide expressed peptides identical to peptide sequences of cell membrane molecules and cytokines involved in regulation of the immune response for development of mAb. In addition, a subsidiary unit of Thermofisher Scientific, Synthetic Biology is being contracted to make bacterial vectors for expressing genes encoding bovine leukocyte membrane molecules for mAb production. The vectors, containing targeted genes encoding membrane molecules, will be used to express the membrane molecules for mAb production. As soon as the mAbs are validated through use and publication, they will be made available through the WSU mAb center http://vmp.vetmed.wsu.edu/resources/monoclonal-antibody-center , Kingfisher Biotech https://www.kingfisherbiotech.com/ , Novus Biologicals https://www.novusbio.com/ and other national and international vendors on request. Novel methods have been developed to facilitate use of the mAbs in study of the immune response ex vivo. The technology will be made available in conjunction with publications describing the new mAbs.
Animal Health Component
100%
Research Effort Categories
Basic
40%
Applied
10%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31533101090100%
Goals / Objectives
Goals: The Wash. State University Monoclonal Antibody Center was established in 1982 to exploit the use of monoclonal antibody (mAb) technology to advance research in the study of the mechanisms regulating the immune response to pathogens and parasites in food and companion animals. Since its initiation, the center has developed mAbs for use in cattle (126), sheep (48), Goats (57), water buffalo (60 anti-bovine mAbs cross reactive with buffalo), pigs (60), llama/alpaca (26), and horse (28). All the mAbs are currently available to the research community through the center: http://vmp.vetmed.wsu.edu/resources/monoclonal-antibody-center , Kingfisher biotech, and Novus Biological. The reagents are also available for re-sale through international distributors on request. The mAbs are also listed through the Veterinary Immune Reagent Network (US VIRN, www.vetimm.org). Many of the advances made in understanding the composition and function of immune systems in the mentioned species have involved the use of the reagents made available through the center.The current goal is to make use of the USDA NIFA grant to continue our efforts to develop mAb reagents critically needed to elucidate the mechanisms regulating the immune response to infectious agents and parasites in cattle. The endeavor will include development of mAbs to phylogenetically conserved epitopes expressed on orthologues of leukocyte differentiation molecules, cytokines, and chemokines to increase the utility of the mAbs for use in comparative analysis of the immune response in cattle and other ruminants. The additional reagents will increase our ability to conduct in depth studies on the immune response to pathogens and vaccines.Objectives: Cattle are one of the major livestock species used as a source of food and dairy products worldwide. Water buffalo, sheep and goats are also important livestock species. Many of the disease problems affecting livestock include these species. Understanding the mechanisms regulating the immune response to the pathogens is essential for developing a rational approach to develop vaccines for the respective pathogens. We have made most of the mAbs needed to identify the lymphocyte cell subsets involved in immune responses in the mentioned species. Our overall objective now is to develop mAbs to fill in the gaps in reagents needed to fully dissect the immune responses to pathogens and validate the functional efficacy of candidate vaccines under development. MAb development will focus on cattle as the target species. 1) Where possible conserved regions of cell membrane molecules will be used to develop mAbs. Development of mAbs to conserved epitopes will help expand opportunities for research to the other important ruminant species. 2) Optimize and make available to the research community, assays we have developed to study antigen processing and presentation by antigen presenting cells in vitro. 3) Make available, in vitro assays we have developed to directly characterize the functional activity of effector T cells proliferating in response to antigens processed and presented by antigen presenting cells. This breakthrough technology affords opportunity to obtain information on the potential efficacy of vaccines under development before testing in the field.
Project Methods
The focus is on making mAbs to membrane molecules involved in regulating the immune response. These include mAbs specific for CD83, CD95, CD152, CD154, CD178, CD192, CD195, CD196, CD197, CD273, CD274, CD279, CD223, CD282, CD284, CD366, IL-22, IL-12R, IL-23R.New England Peptides is a company currently being used to make peptide sequences corresponding to conserved sequences on bovine and other ruminant species chemokine receptors involved in immune regulation. They will be used to develop peptides to chemokine receptors listed in the table of the proposal for mAb development. Through collaborative arrangements, Kingfisher Biotech (as mentioned in the proposal) will provide expressed peptides identical to peptide sequences of cell membrane molecules and cytokines involved in regulation of the immune response for development of mAb. In addition, a subsidiary unit of Thermofisher Scientific, Synthetic Biology is being contracted to make bacterial vectors for expressing genes encoding bovine leukocyte membrane molecules for mAb production. The vectors, containing targeted genes encoding membrane molecules, will be used to express the membrane molecules for mAb production. As soon as the mAbs are validated through use and publication, they will be made available through the WSU mAb center http://vmp.vetmed.wsu.edu/resources/monoclonal-antibody-center , Kingfisher Biotech https://www.kingfisherbiotech.com/ , Novus Biologicals https://www.novusbio.com/ and other national and international vendors on request. Novel methods have been developed to facilitate use of the mAbs in study of the immune response ex vivo. The technology will be made available in conjunction with publications describing the new mAbs.

Progress 03/15/19 to 03/14/20

Outputs
Target Audience:The objectiveproposed to initiate the current project on development of monoclonal antibodies (mAb) to bovine leukocyte differentiation molecules (LDM) was to express LDM and cytokines encoded by CD83, CD95, CD152, CD154, CD178, CD192, CD197, CD273, CD274, CD279, CD223, CD282, CD284, CD366, IL-22, IL-12R, IL-23R. CD95, CD152, CD154, CD178, CD279, CD282, IL-22, IL12R, and IL-23R were expressed for mAb production. mAbs were successfully developed against CD152 and IL-22. In addition, mAbs were successfully developed against molecules not on the list proposed to initiate the project: CD27, TNF-a, IL-6, IL-8 andIL-10. Documentation of specificity of the mAbs is in progress. Manuscripts will be prepared for publication as soon as documentation studies are complete. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training in mAb development, flow cytometry, and analysis of the immune response was provided to 3 post doctoral associates. How have the results been disseminated to communities of interest?As reported above, methodology and use of mAbs developed for research in cattle have been publised. What do you plan to do during the next reporting period to accomplish the goals?As mentioned above, genes encodingmolecutes of interest for developing mAbs have been expressed. These molecules will be used to immunize mice for mAb development. Further training of post doctoral associates will be continued focused on gaining expertese in analysis of the immune response to pathogens.

Impacts
What was accomplished under these goals? Under objective 1, we proposed to initiate the current project on development of monoclonal antibodies (mAb) to bovine leukocyte differentiation molecules (LDM) starting with expression ofLDM and cytokines encoded by CD83, CD95, CD152, CD154, CD178, CD192, CD197, CD273, CD274, CD279, CD223, CD282, CD284, CD366, IL-22, IL-12R, IL-23R. CD95, CD152, CD154, CD178, CD279, CD282, IL-22, IL12R, and IL-23R were expressed for mAb production. mAbs were successfully developed against CD152 and IL-22. In addition, mAbs were successfully developed against molecules not on the list proposed to initiate the project: CD27, TNF-a, IL-6, IL-8 andIL-10. Documentation of specificity of the mAbs is in progress. Manuscripts will be prepared for publication as soon as documentation studies are completed. Under objectives 2 and 3: We have developed assays to study the antigen processing by antigen presenting cells and analyisis of the functional activity of CD4 helper and CD8 effector T cells that develop in response to antigen presentation by antigen presenting cells. the methodologies have been described in our recent publications. Abdellrazeq GS, Fry LM, Elnaggar MM, Bannantine JP, Schneider DA, Chamberlin WM, Mahmoud AHA, Park KT, Hulubei V, Davis WC. Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine. Vaccine 2020 doi: 10.1016/j.vaccine.2019.12.052 Franceschi V, Mahmoud AH, Abdellrazeq GS, Tebaldi G, Macchi F, Russo L, Fry LM, Elnaggar MM, Bannantine JP, Park KT, Hulubei V, Cavirani S, Davis WC, Donofrio G. Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells. Front Immunol 2019;10:2859. doi: 10.3389/fimmu.2019.02859 Abdellrazeq GS, Elnaggar MM, Bannantine JP, Schneider DA, Souza CD, Hwang J, Mahmoud AHA, Hulubei V, Fry LM, Park KT, Davis WC. A peptide-based vaccine for Mycobacterium avium subspecies paratuberculosis. Vaccine 2019;37:2783-2790. doi: 10.1016/j.vaccine.2019.04.040

Publications