Progress 07/01/23 to 06/30/24
Outputs Target Audience:Efforts during this project period were translational research-based and focused on assessing the safety, immunogenicity and efficacy of a staphylococcal bovine mastitis vaccine in a small clinical trial. Specifically during this period, we reached out to stake-holders within the dairy industry as well as experts in veterinary vaccine development as potential investors and collaborators. The long term goals of these efforts will support the dairy industry by prevention of mastitis caused by this highly significant pathogen, and thus the main target audience is the veterinarian and the dairy farmer. Translation of this research also has the potential to improve general public health through improved milk production and reduction in antibiotic dependence. Changes/Problems:A no-cost extension was requested in August of 2024. We were not able to secure the funds through Pentamer Biologics to Boise State for this STTR subcontract until early September 2023through the USDA, so the project was initiated in the fall of 2023instead of the original start date of July 1. The extension has allowed the completion ofall sample collection, data analysis, preparation ofmanuscripts, and preparationfor a Phase II STTR application. What opportunities for training and professional development has the project provided?Post-doctoral training: Elise Overgaard, Ph.D. was supported during this project period. Dr. Overgaard was the main coordinator of the clinical trial (Trial 4, Objective 2) and worked directly with the subcontractor, Johnson Research, LLC (Dr. Nick van Engen) to complete vaccination and challenge of animals, including significant input into trial safety assessment and sampling protocols Dr. Overgaard also purified and tested the vaccine as well as worked collaboratively to complete microbial culture, ELISA clinical analysis, and blood and milk sampling and shipment. Graduate student training: Haley Bridgewater (Ph.D. candidate in Biomolecular Sciences) was supported on this project during this project period. Haley Bridgewater was also involved in all aspects of the bovine trial during this period (Objective 2). She helped to produce and assess the purity of the vaccines, designed T-cell proliferation assays and worked with the Flow Cytometry core at Boise State to complete cellular immunogenicity assays. Haley also worked collaboratively with Dr. Overgaard to complete microbial culture, ELISA clinical analysis, and blood and milk sampling and shipment. Two Boise State Biology undergraduate student volunteers; Sam Billingsley and Alex Soria, also were trained on S. aureus milk plating and CFU determination for bacteriological assays. They completed this work over 3-4 weeks during the project period. Professional development: the USDA collaboration with LARTA and our meetings with our advisor, Tony Simpson, available during this project period enabled training and professional development in product and business development for the PI (JK Tinker), CEO of Pentamer Biologics (Bryan Allinson) as well as Dr. Overgaard and Haley Bridgewater. How have the results been disseminated to communities of interest?We have submitted one publication to the Journal of Dairy Science: Scarbrough, D., Misra, N., Bridgewater, H., Walgren, A., and J. Tinker. Surfaceome and transcriptome profiling to identify Staphylococcus aureus surface antigens expressed in milk. J. Dairy Science. Submitted Oct 2024. This submission is under review We have also submitted an abstract to the Conference of Research Workers in Animal Diseases: Overgaard, E., Bridgewater, H., van Engen, N., and J.K. Tinker. Intramammary cholera-toxin based vaccine with subcutaneous boost shows safety, immunogenicity. Conference of Research Workers in Animal Diseases. Chicago, IL, Jan 18-21.Dr. Overgaard will present this poster in January 2025 Haley Bridgewater successfully defended her thesis: Bridgewater, H. 2025. A Multi-Tiered Approach to Improving Vaccination: Adjuvanticity, Alternate Routes of Delivery, and Science Communication. Ph.D. dissertation, Biomolecular Graduate Program, Boise State University.This thesis work, which contains a chapter on this USDA funded project, was presented as a seminar in December 2024 and will be published in 2025. We will submit a manuscript to the journal Vaccine in January or February 2025: Bridgewater, H., Overgaard, E., Bond, L., van Engen, N. and J.K. Tinker. Safety and Immunogenicity of an enterotoxin-based Intramammary Bovine Staphylococcus aureus Vaccine with Subcutaneous Boost (in preparation). In preparation for Phase II, we have also disseminated outcomes to a number of dairy-industry experts and stakeholders, including researchers, veterinarians and farmers to obtain feedback and identify areas of improvement. What do you plan to do during the next reporting period to accomplish the goals?During the last period of the grant we plan to: For objective 1: continue purification of top candidate antigens as CTA2/B chimeras and/or as affimer fusions revise/resubmit the antigen-discovery manuscript as needed submit invention disclosures for patenting of successful antigen-adjuvant fusions and novel expression vectors For objective 2: complete cytokine ELISAs to support flow cytometry analysis of T cell responses submit complied trial data for publication in the journal Vaccine submit a Phase II USDA SBIR application - March 2025
Impacts What was accomplished under these goals?
Objective 1:Construct a rationally designed multivalent vaccine to prevent clinical and subclinicalS. aureusmastitis by incorporating additional targeted antigens. During the project period, we completed the in silico filtering and scoring of a pool of 26 antigens, identified through previous transcriptomics, proteomics and immunoproteomics, based upon:1) antigen prediction,2)immunoinformatics and epitope prediction, and3)genomic antigen conservation. Of the 26 antigens, we have cloned 11 of these antigens into the original CTA2/B vector (pARLDR19) for vaccine expression and purification. Of these clones, 2-3 antigens have shown evidence of proper folding for vaccine purification, these include: IsdB, Hla, and Spa. In addition, we have constructed a new CTA2/B vector containing a 9 amino acid Ser-Gly linker to facilitate correct protein folding (pAJW003). Lastly, we have constructed a vector using a cholera-toxin binding affimer as a novel method of construction of attachment of antigens to CTB using separate antigen-adjuvant purification. Objective 2: Determine vaccine safety and efficacy after intramammary vaccination in a pilot bovine challenge trial. During the project period, we prepared and completed a randomized/blinded challenge trial using six cows vaccinated through the intramammary route, and boosted subcutaneously, during milking. Milk and serum were collected for cytokine, antigen-specific antibody and T-cell proliferation assay. Clinical outcomes, including rectal temperatures, somatic cell counts, adverse events, vaccine-specific reactogenicity, and bacterial shedding were determined to assess vaccine safety, immunogenicity and efficacy. Data was collected and analyzed for statistical significance.
Publications
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2025
Citation:
Bridgewater, H. 2025. A Multi-Tiered Approach to Improving Vaccination: Adjuvanticity, Alternate Routes of Delivery, and Science Communication. Ph.D. dissertation, Biomolecular Graduate Program, Boise State University. Successfully defended December 2024.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2025
Citation:
Overgaard, E., Bridgewater, H., van Engen, N., and J.K. Tinker. Intrammary cholera-toxin based vaccine with subcutaneous boost shows safety, immunogenicity. Conference of Research Workers in Animal Diseases. Chicago, IL, Jan 18-21.
- Type:
Peer Reviewed Journal Articles
Status:
Submitted
Year Published:
2025
Citation:
Scarbrough, D., Misra, N., Bridgewater, H., Walgren, A., and J. Tinker. Surfaceome and transcriptome profiling to identify Staphylococcus aureus surface antigens expressed in milk. J. Dairy Science. Submitted Oct 2024.
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