Source: BOISE STATE UNIVERSITY submitted to NRP
THE BIOTEK CYTATION 3 TO PROMOTE VACCINE DEVELOPMENT FOR BOVINE MASTITIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005561
Grant No.
2015-67016-22976
Cumulative Award Amt.
$47,041.00
Proposal No.
2014-06321
Multistate No.
(N/A)
Project Start Date
Jan 1, 2015
Project End Date
Dec 31, 2015
Grant Year
2015
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
BOISE STATE UNIVERSITY
1910 UNIVERSITY DRIVE
BOISE,ID 83725
Performing Department
Biological Sciences
Non Technical Summary
Infections in the bovine udder represent highly significant economic burdens for the dairy industry worldwide. Increases in antibiotic resistance and the transmission of contagious bacterial agents that cause chronic disease have reinforced the need for effective vaccines. Vaccines have the potential to eliminate clinical and subclinical infection, reduce the dependence on antibiotics and improve overall milk production. We have proposed the characterization of novel vaccines to prevent bovine mastitis caused by the bacteriaStaphylococcus aureus and Streptococcus uberis. These pathogens can go undetected as subclinical disease but have detrimental effects on milk quality and production. Clinical field trials to assay the immunogenicity and protective efficacy of these vaccines have been designed and initiated. To support the laboratory development and translation of this technology, the funds from this equipment grant will support the aquisition of a BioTek Cytation 3 multimode microplate reader with automated fluorescent imaging system (#CYT3MFV, BioTek, Winooski, VT). This system will promote three main objectives to advance vaccine research in our laboratory; 1) to improve the production and quantification of purified vaccine products, 2) to streamline immunoassays to assess vaccine efficacy and 3) to characterize and screen vaccine candidates prior to animal testing. The BioTek Cytation 3 will significantly enhance our efforts to develop an effective vaccine to prevent bovine mastitis, and this research has broad implications for the worldwide dairy industry.
Animal Health Component
30%
Research Effort Categories
Basic
10%
Applied
30%
Developmental
60%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113410109025%
3113410110020%
3114010110010%
3153410110020%
3153410109025%
Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
4010 - Bacteria; 3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;
Goals / Objectives
The major goal of this equipment grant is to obtain state-of-the-art technology that will facilitate vaccine research in our laboratory.To support and streamline our laboratory research with the goal of vaccine development to combat staphylococcal and streptococcal bovine mastitis, we have requested funds to purchase a Cytation3 multimode microplate reader (#CYT3MFV, BioTek, Winooski, VT).The main objectives for research using this equipment are:Objective 1: To quantify purified vaccine products and assay in vitro antigen delivery. This objective centers on the characterization of candidate vaccines prior to in vivo clinical analysis. The BioTek Cytation3 has a monochromator based system for UV-Vis absorbance and pre-programmed nucleic acid and protein quantitation protocols in accompanying software. This function will support current cloning, PCR and protein expression protocols for vaccine production. In addition, the fluorescence microscopy function of the Cytation3 will facilitate direct visualization of cells for in vitro antigen uptake and immune stimulation assays prior to clinical assessment.Objective 2: To streamline immunoassays to assess vaccine efficacy. This objective focuses on the numerous downstream immunoassays necessary to quantify vaccine efficacy during and after clinical trials. These assays include antigen-specific ELISA of serum, milk and bovine nasal secretions as well as cellular proliferation, activation and cytokine induction assays. Currently these time-consuming assays require the use of a departmentally shared microplate reader and flow-cytometer. Acquisition of the Cytation3 will enable rapid quantitation and data analysis within our laboratory. Objective 3: To compare the in vitro efficacy of distinct antigen-adjuvant combinations. An effective S. aureus vaccine to prevent bovine mastitis will require the incorporation of multiple conserved antigens and distinct adjuvants that can induce cellular and humoral responses. The assessment of adjuvant activity in vitro prior to animal testing is a useful screening process to optimize vaccine formulations. These screening assays can include antigen uptake, proliferation, gene expression, cell viability and cytokine analysis. The Cytation3 will facilitate a broad range of cell-based assays for the purpose of basic research or applied drug discovery. We propose the comparison of a number of enterotoxin A2/B chimeras in vitro that we have previously constructed as potential vaccines to prevent bovine mastitis.
Project Methods
Objective 1: To quantify purified vaccine products and assay in vitro antigen delivery. Efforts for this objective will include new and improved protocols for protein and nucleic acid quantitation that will facilitatecloning, PCR and protein expression protocols for vaccine production. In addition, the fluorescence microscopy function of the Cytation3 will enable direct visualization of cells for in vitro antigen uptake and immune stimulation assays prior to clinical assessment. The outcomes of this objective will be evaluated by the successful production and scaling up of new vaccines for animal testing, and will lead to the publication, presentation and/or patenting of combined data. Objective 2: To streamline immunoassays to assess vaccine efficacy. Efforts for this objective will specifically include much improved protocols and methodology forantigen-specific ELISA of serum, milk and bovine nasal secretions as well as cellular proliferation, activation and cytokine induction assays. The outcomes of this objective will similarly be evaluated by successful vaccine trials that will lead to publication, presentation and/or patenting.Objective 3: To compare the in vitro efficacy of distinct antigen-adjuvant combinations. Efforts for this objective willassess adjuvant activity in vitro prior to animal testing to optimize vaccine formulations. These screening assays will include antigen uptake, proliferation, gene expression, cell viability and cytokine analysis. The outcomes of this objective will be the identification of promising new vaccine formulations in vitro that can be tested further in vivo.

Progress 01/01/15 to 12/31/15

Outputs
Target Audience:The target audience for this equipment grant isthe undergraduate, graduate,post-doctoralstudents and research techniciansin the PI's laboratory. During the project period one reseach technician (Tyler Wines), twoPh.D level graduate students (Neha Misra and Nisha Shrestha), threePh.D. rotation students (Sheenah Bryant, Eric Swiecki, Nate Redman), 8undergraduate students (Laura Rogers, Emily Price, Hannah Weaver, Connor Richmond, Dana Clary, Libby Stewart, Kimmy Brown, Kristina Chapman) a high school teacher (Sarah Westcott), a post-doctoral fellow (Farzana Siddique) and two Master's students (Colton Knopp and Sikha Neupane) have been trained on the use of this equipment. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student training: Neha Misra, Ph.D. candidate, was trained on this equipment and has used it extensively. Data obtained has enabledNeha to attend one regional conference (NIH IDea Western Regional Conference) and one localconference (Idaho Academy of Sciences). Colton Knopp, M.A., Biological Sciences, utlized this equipment tocompletehis projectstudies in Dec of 2015. One rotation Ph.D. student; Eric Sweicki, was also trained on experimentationspecific to this project. Undergraduate training: Laura Rogers, Hannah Weaver, Connor Richmond, Elizabeth Stewart,Dana Clary and Kristina Chapmanare Boise State Undergraduates who have benefited fromtrainingon use of this equipment. How have the results been disseminated to communities of interest?The P.I. has disseminated research efforts utilizing this equipment, and data obtained from a concurrent USDA award (Tinker USDA#2013-01189)to the media in the form of a newspaper article on technology transfer (IdahoStatesman, published 12-29-15), and to industry in the form of a presentation to the Idaho Technology Council CapitalConference (Oct 2015). The PI has disseminated this work to the science community through presentations at scientificconferences (Keystone Conference on Veterinary Immunology, Jan 2015, Idaho Academy of Sciences, March 2015, IdahoConference on Undergraduate Research, July 2015 and the NIH Idea Western Regional Conference, Oct 2015) as well asefforts on submission and preparation of manuscripts. The PI continues to work with the Central District Health ImmunizationAdvisory Committee, and is currently teaching a course on Vaccinology with a significant service-learning component in which vaccine information and education is disseminated to local high school and middle school students. 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 this project period we have developedprotocols using the BioTek Cytation 3 to assay antigen delivery into immune cells in vitro to characterize our vaccine delivery plateform prior to animal studies. The flourescent microscopy and cell counting function has also been used to characterize the function of a novel toxin from Salmonella Typhimurium. In addition, we have used the plate reader function for protein assays that are essential for the development of2D gelelectrophoresis. We are using 2D electrophoresis to identify novel S. aureus vaccine candidates (Tinker USDA#2013-01189). An NIH pilot COBRE grant was received to perform 2D gelelectrophorsis on proteins that specifically bind to extracellular matrix molecules. These studies are also currently underway. Inaddition we have completed studies using the BioTek Cytation 3to characterize ECM binding of bovine S. aureus grown in milk and the ability to blockECM binding.Apublicationwas submitted that characterized the expression and variablity of IsdA from bovine milk, and a second manuscript will besubmitted soon on the expression of ECM adhesins from S.aureus grown in milk.Objective 2) during this project period we completed a number of immunoassays on bovine milk and blood samples from abovine vaccine immunogenicity study. This study (Trial 1, USDA Tinker#2013-01189)began on Oct 1, 2014 and was concluded on Dec 17, 2014. 21 animals were vaccinated intranasally and blood,milk, nasal washes and nasal swabs were collected from all cows. Data analysis revealeda significant increase in anti-IsdAand anti-ClfA responses in milk from vaccinated cows and a trend of increased anti-IsdA and anti-ClfA responses in serum.Continuedefforts, and use of the BioTek Cytation 3, are focused on protocols for cellular analysis and implementing Trial #2 with a higherdose and more animals per group.Objective3) during this project period we have used the BioTek DNA quantification to aid in the cloning of novel vectors to express cholera toxin and E.coli heat-labile toxin A2/B fusions. These proteins will be purified and antigen uptake quantified and compared using the flourescent microscopy function of thisinstrument.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Staphylococus aureus IsdA and ClfA- cholera toxin A2/B fusions as mucosal mastitis vaccines. Wines, T.F, Misra, N. and J.K. Tinker 2015. Keystone Conference on Immunity to Veterinary Pathogens:Informing Vaccine Development. January 20, 2015. Keystone, CO.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Regulation of Staphylococcus aureus adhesin gene expression in vitro simulating bovine mastitic conditions. Misra, N. and J.K. Tinker. 2015. NIH IDea Western Regional Conference. Oct 12, 2015. Coeur d'Alene, ID.
  • Type: Other Status: Accepted Year Published: 2015 Citation: The effect of a novel vaccine on Staphylococcus aureus binding and colony formation. Knoppm C.J. A project paper for the Masters in Arts in Biological Sciences, Boise State University. Dec 2015.
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Characterization of Staphylocccus aureus IsdA from Bovine isolates. Wines, T.F., Misra, N., Williams, J.E., McGuire, M.A. and J.K. Tinker. 2015. Veterinary Microbiology. Submitted Aug 2015, rejected Oct 2015. To be re-submitted to mBIO March 2016.