Progress 01/01/14 to 12/31/18
Outputs
Target Audience:Efforts during this final project period were research based and focused on completing trialsand analysis of an experimental bovine vaccine for mastitis. The long term goals of these efforts will support the dairy industry by prevention of mastitis caused by this significant pathogen, and thus the main target audience is the dairy farmer and the animal vaccine industry. Translation of this research also has the potential to improve general public health through improved milk production and reduction in antibiotic dependence. Specifically during this project period a number of undergraduate students at Boise State University with diverse backgrounds (Aurora Vogel, Jim Schroeder, Gary Dunn, Edgardo Alyala Tapia, Nateijiee Truman, Christian Johnson, Tressa Rudd, Omid Mohammad Mousa) were supported and trained in research on this project. In addition two graduate students at Boise State (Danielle Holt and Elise Overgaard) and one post-doctoral researcher at the University of Idaho (Hussain Alabdullah) were supported by this project in 2018. Changes/Problems:During Trial 3 of final project period (Objective 3) one animal was euthanized during the study due to systemic bacterial infection immediately post-challenge. This was a vaccinated animal. The other animals did not show any signs of systemic infection and thus the trial was continued with 3 vaccinated and 3 control animals. Subsequent microbiological, clinical, and post-mortem investigation indicated that this animal had an undetected E.coli intramammary infection prior to S. aureus challenge which became systemic and likely contributed to the animal's rapid decline. In addition, despite the injection of a lower CFU of S. aureus Newbould 305 than originally proposed based upon previous studies, bacterial shedding was very high in all animals throughout most of the challenge period and treatment was longer and more difficult than anticipated. The knowledge gained regarding challenge methodology from this final study will greatly improve the design of S. aureus challenge models and future trials. What opportunities for training and professional development has the project provided?Post-doctoral training: Hussain Alabdullah, Ph.D. was supported during the final project period through the subcontract with the Co-PI (Dr. Mark McGuire, University of Idaho). Dr. Alabdullah was key to coordinating Trial 3 (Objective 3), as well as providing animal support, microbial culture, clinical analysis, and blood and milk sampling and shipment. Graduate student training: Danielle Scarbrough (M.S. candidate in Biomolecular Sciences) and Elise Overgaard (M.S. candidate in Biomolecular Sciences) have been supported on this project during the final period. Danielle Scarbrough, began her studies in the fall of 2017 and will continue efforts started by a former Ph.D. student to characterize and identify new S. aureus vaccine antigens. She was supported by this grant and Simplot Industries. Danielle's work on this project was also supported by a small bioinformatics grant (Boise State COBRE/INBRE) that enabled her to go to a 4 day Bioinformatics workshop (New Mexico INBRE Differential Gene Expression Workshop, Santa Fe, NM Nov 12-16, 2018) to enhance her thesis project. Elise Overgaard started her M.S. in the fall of 2018 and has been supported by this funding as well as funding through Boise State Graduate Studies. Elise has been working on immunoanalysis from Objective 3, and has applied for the Ph.D. program for the fall of 2019. Three undergraduate research assistants have been supported directly on this grant during this project period and they include: Omid Mohammad Mousa, Gary Dunn, and Edgardo Ayala Tapia. Omid will apply for the Biomolecular Ph.D. program in 2020, Gary has entered the Microbiology Ph.D. program at Montana State University (Fall of 2018) and Edgardo has applied to M.S. programs in Microbiology for the fall of 2019. In addition a number of fellows, interns and undergraduate volunteers have contributed to these studies and they include: Hunter Johnson, Nateijie Truman, Aurora Thomson, Jim Schroeder, Tressa Rudd and Adriana Rodriguez. Many of these students remain in the Tinker Laboratory at Boise State. How have the results been disseminated to communities of interest?During the final project period we have published two manuscripts on this work: https://www.sciencedirect.com/science/article/pii/S0022030218304053?via%3Dihub https://www.sciencedirect.com/science/article/pii/S0264410X18305644?via%3Dihub The P.D. has also disseminated this research in the form of poster presentations to conferences during this final project period, which include: the Boise State Undergraduate Research Conference (2 posters, Boise, ID, April 2018), the Idaho Conference on Undergraduate Research (2 posters, Boise, ID, July 2018), and the Idaho INBRE Biomolecular conference (2 posters, Coeur d'Alene, ID, August 2018). The P.D. also presented a talk on this research at the Treasure Valley COBRE/INBRE meeting in Boise (March 20, 2018) and at the annual CRWAD/USDA PD conference in Chicago (Dec. 3, 2018). The PD continues to work with the Central District Health Immunization Advisory Committee, teaches Vaccinology, and Veterinary Immunology and Microbiology courses and collaborates with local veterinarians (Dr. Brian Mitchell, Dr. Stuart Shoemaker and Dr. Matt Edmonds), and also maintains a long-standing collaboration with the University of Idaho (Dr. Mark McGuire) and Washington State University (Dr. Larry Fox). Overall number counts for entire project period: 3 peer-reviewed publications published (plus 2 in preparation) 1 post-doctoral fellow trained 5 graduate students trained (1 Ph.D.4 M.S., plus 3 Ph.D. rotation students) 17 undergraduates trained 18 poster/oral conference presentations 1 Ph.D. thesis published 3 patent continuations/amendments filed,2 of which are issued 3 connections with industry pursued, currently no patent licensing 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) Rigorously characterize immune responses stimulated by intranasal, intramammary and intravaginal administration of A2/B chimeras containing S. aureus IsdA and ClfA in cows. Complete and reported in year 2017. Percent completion of Objective 1 = 100%. Objective 2) Utilitize genomic and proteomic analysis to identify conserved antigens for incorporation into A2/B chimeras. Specific goals under Objective 2 include: staphylococcal strain collection and genomic analysis, immunoproteome assessment and construction of novel chimeras, including those for Streptococcus uberis. In 2017 (previous periwe published results from our genetic analysis of 40 bovine staphylococcal isolates for the presence and conservation of isdA and other key virulence factors (Misra et al., 2017 FEMS Micro Lett). During 2018 we published results from 2D seroproteomic studies for the identification of immunoreactive S. aureus proteins (Misra et al. 2018 Journal of Dairy Science). During this project period we continued the development of cholera toxin CTA2/B chimeras containing S. uberis antigens, and were successful in the purification of a S. uberis SUAM-CTA2/B chimeric vaccine. These studies will be followed up in the future through potential collaborations with other USDA scientists (Dr. Stephen Oliver, University of Tennessee). A small local grant has supported the continuation of S. aureus antigen discovery through surfaceome and transcriptomic analysis (RNAseq) of cultures grown in milk. It is expected that a manuscript on this work will be submitted in 2019. Percent completion of Objective 2 = 100%. Objective 3) Evaluate the protective efficacy of IsdA-CTA2/B and ClfA-CTA2/B in a bovine model of staphylococcal mastitis. Specific goals under Objective 3 include: bovine immunization and challenge, sampling and monitoring and CFU and immune analysis. This final bovine clinical trial (Trial 3) began in June of 2018 and was completed by the end of July 2018. Trial 3 was performed at the University of Idaho College of Agriculture and Life Sciences Dairy Center under the supervision of the Co-PI; Dr. Mark McGuire. The study utilized a total of 7 cows and assessed the ability of two intranasal doses (total of 1200 mg) of IsdA-CTA2/B and ClfA-CTA2/B to protect cows from intramammary challenge with isogenic S. aureus mastitis. Animals were vaccinated through the intranasal route on days 1 and 14 and challenged in two quarters with 400 CFU of S. aureus Newbould 305. Animals were followed for 10 days post-challenge prior to antibiotic treatment and release. Results from this trial indicate that vaccination promoted reduced bacterial shedding in milk toward the end of the 10-day challenge period and that this reduction coincided with a significant decrease in somatic cell count (SCC) and slight reduction in clinical severity in vaccinated animals. Antibody analysis of milk is still underway, but antigen-specific S. aureus serum IgG responses have been detected during the challenge period using ELISA and opsonophagocytosis. Experimentation proposed for this objective is complete, but data analysis and compilation is still underway. Work is expected to be submitted for publication in 2019. Percent completion of Objective 3 = 100%. Overall Impacts: The availability of an effective S. aureus vaccine to prevent bovine mastitis would have multiple positive impacts on the dairy industry. These include: 1) improved animal health, 2) improved milk production, 3) reduction in herd somatic cell count (SCC), and 4) reduction in animal loss, veterinary costs, and antibiotic use. These benefits would translate to increased food production and efficiency, and may also contribute to human health through improved nutrition and a reduction of antibiotic use. The vaccine described and evaluated in these studies is distinctive from other experimental S. aureus vaccines as: 1) it is a purified subunit protein vaccine, 2) it targets two highly conserved S. aureus antigens involved in bacterial virulence, and 3) it can be delivered via the nose, with potential for other routes of delivery including the mouth, skin, vagina and mammary gland. Use of these alternative routes will promote targeted immune responses to prevent udder colonization, and will also reduce transmission of needle-born disease in herds and negative effects from needles on meat quality. Results from field trials in Objective 1 indicated that the vaccine is immunogenic in dairy cows and safe after intranasal delivery. Results from Objective 2 provided evidence that the antigens in the current vaccine (IsdA and ClfA) are present and conserved across many different bovine S. aureus isolates. In addition this objective opened up new avenues for research into previously uncharacterized bovine S. aureus candidate antigens and enabled construction of a novel S. uberis vaccine. Lastly, Objective 3 has provided a capstone efficacy analysis; indicating, in small scale, that the vaccine reduces shedding and clinical outcomes of S. aureus from the bovine udder. These studies, which would not have been possible without USDA-NIFA support, have provided an essential foundation for future collaborations that will enable large-scale clinical trial and manufacture of this novel vaccine. Lessons learned will also improve future S. aureus vaccine study in bovines, and support the exploration of CTA2/B chimeras as vaccines to prevent other mastitis pathogens, such as S. uberis, S. agalactiae and E.coli. Long term goals for this research program include a potential universal mastitis vaccine that would have very broad and positive impacts on the U.S. and global dairy industry.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Misra, N, Wines, T.F., Knopp, C.L. Hermann, R., Bond, L., Mitchell, B.,
McGuire, M. and J.K. Tinker. 2018. Immunogenicity of a Staphylococcus aureus-cholera toxin A2/B vaccine for bovine mastitis. Vaccine 36(24):3513-3521.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Bacterial quantification from a bovine challenge study to determine vaccine
efficacy against S. aureus mastitis. Schoeder, J., Dunn, G., Williams, J.,
Alabdullah, H., McGuire, M., and J. Tinker. Idaho INBRE annual conference, Moscow, ID., July 31, 2018 (poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Exploring the ideal excipients for a chimeric vaccine against bovine mastitis. Mousa, O.M. and J.K. Tinker. Boise State University Undergraduate Research Conference. April 4 2018. Boise, ID (poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Forming a bovine mastitis vaccine using S. aureus IsdH and cholera toxin. Truman, N. and J.K. Tinker. Boise State University Undergraduate Research Conference. April 4 2018. Boise, ID (poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
A vaccine to prevent Staphylococcus aureus in bovines. J.K. Tinker. Treasure Valley INBRE/COBRE conference. March 20, 2018 (oral).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
A Staphylococcus aureus challenge trial to assess the efficacy of an enterotoxin-based vaccine for bovine mastitis.J.K. Tinker. CRWAD and USDA NIFA PD conference. Dec 3, 2018. Chicago, IL (oral).
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Misra, N., Pu, S., Holt, D., M. McGuire and J.K. Tinker. 2018.
Immunoproteomics to identify Staphylococcus aureus antigens
expressed in bovine milk during mastitis. J. Dairy Science 101(7) 6296-6309.
|
Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Efforts during this project period were research based and focused on; 1) completing experimentation and analysis of a second vaccine immunogenicity trial in bovines, 2) using seroproteomics to identify new S. aureus vaccine antigens that are immunogenic in mastitic cows, 3) publication of these efforts. The long term goals of these efforts will support the dairy industry by prevention of mastitis caused by this significant pathogen, and thus the main target audience is the dairy farmer and the animal vaccine industry. Translation of this research also has the potential to improve general public health through improved milk production and reduction in antibiotic dependence. During this project period we have also continued collaborations with local veterinarians and outreach to industry to aid in product translation. Changes/Problems:A major change during this project period was the need to reduce the number of cows used in the proposed challenge study (Objective 3). This study has now been designed as a pilot study with an n of 4 cows (total of 7). Statistical analysis determined that an n of 15 cows per group was necessary to obtain statistical significance. Consultation with Dr. McGuire determined that this was not possible with budget constraints. Thus we are moving forward with a smaller proposed study to obtain essential information to continue to move the vaccine efforts forward and for future challenge studies. What opportunities for training and professional development has the project provided?Graduate student training: Neha Misra, Ph.D. candidate, has been supported as a Research Assistant on this project for two years, which ended in the fall of 2016. Neha was supported by the Biomolecular Ph.D. Program during the spring and summer of 2017, and defended her thesis successfully in the October of 2017. Her thesis work was entirely supported by this project. She will officially graduate in December 2017. During this project period Neha was invited to give an oral presentation at the 11thVaccine Congress in San Diego, CA (Sept 2017). One rotation Ph.D. student; Maranda Cantrell, was trained on experimentation specific to this project in the fall of 2017. One MS student in the Biomolecular Graduate program, Danielle Holt, began her studies in the fall of 2017 and will continue efforts started by Neha Misra to characterize S. aureus antigens. She is supported currently by this grant and also Simplot Industries. Four undergraduate research assistants have been supported during this project period, either directly or indirectly on this grant, and include: Kristina Chapman, Omid Mousa, Gary Dunn, and Orion Thompson-Vogul (awarded an INBRE summer fellowship, summer 2017). How have the results been disseminated to communities of interest?In addition to publication (Misra et al, 2017 FEMS Micro Lett), we have also submitted two manuscripts during the project period (Misra et al, Vaccine and Misra et al J. Diary Science). The P.D. has also disseminated this research in the form of presentations to conferences during the project period, which include: the Boise State University Graduate Conference (Boise, ID March 2017), the Boise State Undergraduate Research Conference (Boise, ID, April 2017), the Idaho Conference on Undergraduate Research (Boise, ID, July 2017), the Idaho INBRE Biomolecular conference (Coeur d'Alene, ID, August 2017), and the 11th Vaccine Congress in San Diego, CA (Sept 2017). The P.D. has also been in connection with potential industry partners and has presented this as a research talk to representatives from the animal divisions of Merck and Eli Lily during 2017. The PD continues to work with the Central District Health Immunization Advisory Committee, teaches Vaccinology, and Veterinary Immunology and Microbiology courses and collaborates with local veterinarians as well as maintains a long-standing collaboration with the University of Idaho (Dr. Mark McGuire) and Washington State University (Dr. Larry Fox). What do you plan to do during the next reporting period to accomplish the goals?Objective 1) Rigorously characterize immune responses stimulated by intranasal, intramammary and intravaginal administration of A2/B chimeras containing S. aureus IsdA and ClfA in cows. None: completed. Objective 2) Utilitize genomic and proteomic analysis to identify conserved antigens for incorporation into A2/B chimeras. For this objective we will: 1) continue experimentation on IsdC and EsxA, as well as other identified S. aureus antigens, to determine immunogenicity, in vivo expression, and sequence conservation from bovine isolates, 2) incorporate newly identified S. aureus antigens into a CTA2/B mucosal mastitis vaccine, 3) continue efforts to express and purify Streptococcus uberis CTA2/B chimeric vaccines. Objective 3) Evaluate the protective efficacy of IsdA-CTA2/B and ClfA-CTA2/B in a bovine model of staphylococcal mastitis. For this objective we will: 1) implement Aim 3 challenge studies in collaboration with Dr. McGuire (University of Idaho), 2) compile and submit results for publication. During this period we will also continue to work with the Boise State Office of Technology Transfer to obtain industry partners and identify new collaborations to advance these efforts to make an effective vaccine to prevent bovine mastitis.
Impacts
What was accomplished under these goals?
Mastitis is one of the most important diseases affecting dairy cattle worldwide, with an estimated economic impact of $1.7-2 billion annually in the U.S (National Mastitis Council, Madison, WI 1996). Staphylococcus aureus and Streptococcus uberis are important bacterial agents of mastitis that can produce chronic and subclinical disease. This infection is difficult to detect and spreads rapidly within herds. Despite improved control measures over the past two decades, S. aureus and S. uberis remain leading agents of mastitis and cause considerable economic loss every year in the U.S. To more effectively prevent disease and reduce dependence on antibiotics, the ideal method of mastitis control is a vaccine. The delivery of vaccines by the mucosal route, such as intranasal, or directly to the mammary gland, has the potential to be a more effective and less costly alternative to the use of needles. To construct a needle-free S. aureus mastitis vaccine, we have utilized Cholera Toxin (CT) as a vaccine adjuvant, or immune-helper molecule. CT is a potent modulator of immune responses that is known to promote immunity from mucosal surfaces. Chimeric CTA2/B molecules comprise the CT binding subunit and A2 domain fused to a vaccine antigen. These molecules are non-toxic, easy to purify and associate the vaccine antigen directly to the adjuvant. It is our objective to characterize the efficacy of these purified molecules as vaccines against S. aureus bovine mastitis, and to construct additional A2/B chimeras to incorporate into a multivalent combination S. aureus and S. uberis vaccine. Objective 1) Rigorously characterize immune responses stimulated by intranasal, intramammary and intravaginal administration of A2/B chimeras containing S. aureus IsdA and ClfA in cows. Specific goals under this objective include: vaccine production and immunization, bovine sampling and monitoring, immune assays and data analysis. During this project period we completed all immunoassays and data analysis for a second immunogenicity trial (Trial 2) to assess responses to a mucosal bovine vaccine. This vaccine contains the two S. aureus adhesins, IsdA and ClfA. This trial was distinct from the first trial (Trial 1, completed in 2015) in that: a) the sample size was increased from 7 per group to 11 cows per group, b) the concentration of total antigen delivered was increased from 900ug to 1200ug in a total of two doses instead of three doses, and c) more samplings of blood were taken to assess immune responses and to collect cells for flow cytometry. The vaccine was delivered intranasally, as in Trial 1. Data from Trial 2 include: 1) Anti-IsdA milk and serum humoral responses, 2) Anti-ClfA milk and serum humoral responses, 3) Anti-IsdA and ClfA cellular proliferation responses, 4) cytokine stimulation analysis, 5) antigen-specific opsonophagocytic assays from serum and 6) antigen-specific opsonophagocytic assays from milk. ELISA data from Trial 2 is supportive of Trial 1, but indicates that a three-dose schedule, even with lower vaccine concentrations, may be optimal for immune responses upon freshening. Cellular responses from Trial 2 indicated a mixed Th2-Th1 responses with trend to Th2. Opsonophagocytic assays are very promising, showing antigen -specific antibody activity in both serum and milk all the way up to day 60. Results from Trial 2 have been combined with ELISA analysis from Trial 1 and submitted to Vaccine in October 2017 for publication. Percent completion of Objective 1 = 100%. Objective 2) Utilitize genomic and proteomic analysis to identify conserved antigens for incorporation into A2/B chimeras. Specific goals under objective 2 include: staphylococcal strain collection and analysis, proteome analysis and construction of novel chimeras. During 2017 we published results from our analysis of a collection of over 40 strains of bovine Staphylocccus for the presence and conservation of the IsdA antigen (Misra et al., FEMS Micro Lett). This report is a comprehensive assessment of IsdA as a bovine vaccine antigen and includes: PCR analysis, RT-PCR analysis, sequence alignment, anti-IsdA antibody activity, and assessment of IsdA isoform antibody reactivity and fibronectin-binding activity. Results indicate that the presence of isdA is strongly conserved in bovine isolates, but that there are at least two common variants of this protein that affect immune-reactivity and function. Also, within this aim, we have made substantial progress toward the identification of new surface antigens from bovine S. aureus that are immunogenic in mastitic cows. This work has used a combined seroproteomics approach that incorporates 2D gel electrophoresis, immunoblotting, mass spectrometry and proteomics. Using this approach we have identified 8 S. aureus surface adhesins and/or virulence factors that represent new antigens to be characterized for a bovine vaccine. Two of these antigens, IsdC and EsxA, we have purified and characterized for immune-reactivity using infected bovine milk. isdC and esxA have also been determined to be present in a majority of bovine isolates and have low sequence variation. This work was submitted in October 2017 to The Journal of Dairy Science for publication. We have also continued our efforts to construct cholera toxin CTA2/B chimeras containing the Streptococcus uberis antigens SUAM and MtuA, as well as a new uncharacterized adhesion, 111234A. Future efforts in this aim will include identification of additional S. uberis antigens, as well as further characterization of the S. aureus antigens and incorporation into CTA2/B chimeras for vaccine development. Percent completion of Objective 2 = 80%. Objective 3) Evaluate the protective efficacy of IsdA-CTA2/B and ClfA-CTA2/B in a bovine model of staphylococcal mastitis. Specific goals under objective 3 include: bovine immunization and challenge, sampling and monitoring and CFU and immune analysis. An IACUC protocol has been constructed for challenge studies, and is currently in progress through the Boise State and University of Idaho committees. IBC review has been approved for these studies and a no-cost extension has been approved on the grant to complete this work. This final trial (Trial 3) will utilize a total of 7 cows and will assess the ability of two intranasal doses (total of 1200 ug) of IsdA-CTA2/B and ClfA-CTA2/B to protect cows from challenge with isogenic S. aureus mastitis. The trial will take a total of 60 days and will occur in the spring and early summer of 2018 in collaboration with Dr. Mark McGuire of the University of Idaho. Percent completion of Objective 3 = 20%. Overall Impacts: The development of an effective S. aureus vaccine to prevent bovine mastitis would have direct impacts in the dairy industry. These include: 1) improved animal health, 2) improved milk production, 3) reduction in herd somatic cell count (SCC), and 4) reduction in animal loss, veterinary costs, and antibiotic use. These benefits would translate to increased food production and may also contribute to human health through improved nutrition and reduction antibiotic use. The delivery of vaccines to cows via the nose, mouth or udderwill induce immune responses to prevent colonization, reduce transmission of needle-born disease in herds and reduce negative effects from needles on meat quality. In addition, these studies have identified S. aureus antigens that are involved bovine colonization of the udder, expressed during infection, and highly conserved in bovine strains. These efforts are essential to promote vaccine efficacy. Lastly, lessons learned from these studies on the use of CTA2/B chimeras as vaccines to prevent S. aureus will be used to construct vaccines against other mastitis pathogens such as S. uberis, S. agalactiae and E.coli that would have very broad impacts on the dairy industry.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Misra, N., Wines, T.F., Knopp, C.L., McGuire,M. and J.K. Tinker*. 2017.
Expression, immunogenicty and variation of IsdA from bovine isolates of
Staphylococcus aureus. FEMS Micro Lett. 364(9):fnx082
*corresponding author
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Misra, N, Wines, T.F., Knopp, C.L. Hermann, R., Bond, L., Mitchell, B.,
McGuire, M. and J.K. Tinker*. Immunogenicity of a mucosal Staphylococcus aureus vaccine for bovine mastits. Vaccine. Submitted Oct 2, 2017.
*corresponding author
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Misra, N., Pu, S. M. McGuire and J.K. Tinker*. Immunoproteomics to identify
Staphylococcus aurues antigens expressed during mastitis. J. Dairy Science. Submitted Oct. 24, 2017.
*corresponding author
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Targeting bacterial extracellular-matrix interactions to construct a mucosal
vaccine for Staphylococcus aureus bovine mastitis. Misra N*. and J.K Tinker. 11th Vaccine Congress, San Diego CA. Oct 2017 (Oral).
*presenting author
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2017
Citation:
Development of a cholera toxin CTA2/B based Staphylococcus aureus vaccine to prevent bovine mastitis. Neha Misra, successfully defended Oct 24, 2017.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Expression and purification of Staphylococcus aureus vaccine antigens to construct a bovine mastitis vaccine. Thompson-Vogel O. and J.K. Tinker. Idaho INBRE Annual Research Conference, Moscow, ID Aug 8, 2017 (winner, second place for undergraduate poster presentation).
|
Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Efforts during this project period were research based and focused on;1) completing a second Staphylococcus aureus vaccine trial in a large local commercial dairy farm to assess immunogenicity, 2) identifyingnewS. aureus vaccine antigens that are immunogenic in mastitic cows, 3) characterizing the variability and expression of the S. aureus IsdA vaccine antigen, and 4) constructing a Streptococcus uberis chimeric vaccine. The long term goals of these efforts will support the dairy industry by prevention of mastitis caused by these significant pathogens, and thus the main target audience is the dairy farmer. Translation of this research also hasthe potential to improve general public health through improved milk production and reduction in antibiotic dependence. This year we have also continued collaborations with local veterinarians and outreach to industry to aid in product translation. Changes/Problems:One major change in specific Aim 1 has been the inability to pursue alternative routes of administration of the IsdA and ClfA-CTA2/Bvaccine. We performed intitial trials (Trial 1 and 2) only using the intranasal route, and will complete challenge studies using this route. While we believe other routes of administration hold promise (intravaginal, intramamary), potential safety concerns about delivering a new vaccine via these untested routes have prevented us from attempting these studies, as intially proposed, in a commercial farm. These studies will have to be performed in the future under more controlled conditions in collaboration with the University of Idaho. What opportunities for training and professional development has the project provided?Graduate student training: Neha Misra, Ph.D. candidate, has been supported as a Research Assistant on this project for two years, which ended in the fall of 2016. Neha is currently supported by the Biomolecular Ph.D. Program and is expected to graduate in December of2017. This project period Neha attended one international conference (ISV, Boston, MA) and presented her work in an oral short talk as well as a poster, and was awarded a competitive travel grant ($500). Colton Knopp, M.A. candidate, began work on this project in the fall of 2014 and completed his studies in December 2015. Colton was able to continue efforts, supported by the Office of Sponsored Programs, through the summer of 2016. One rotation Ph.D. student; Rosey Whiting, was trained on experimentation specific to this project in the fall of 2016. Three undergraduate research assistants have been supported during this project period and include: Hannah Weaver, Connor Richmond and Kristina Chapman. In addition a number of undergraduate fellows and volunteer undergraduates have been trained on research related to this project, including: Kimmy Brown, Kate Gleason, Orion Thompson-Vogul, Sofiya Spencer, Eli Luna, and Matt Dobrowski. How have the results been disseminated to communities of interest?The P.D. has desseminated this research in the form of presentations to conferences during the project period, which include: the ASM Intermountain Branch annual conference (Salt Lake City, UT), the Idaho INBRE Biomolecular conference (Coeurd'Alene, ID), the Idaho Conference on Undergraduate Research (Boise, ID), and the annual International Society for Vaccines (ISV) in Boston, MA. In addition, the P.D. has presented this as aresearch talk at a the local Treasure Valley INBRE/COBRE meeting (Boise, ID) in May 2016. THe PD continues to work with the Central District Health Immunization Advisory Committee, teaches Vaccinology, and Veterinary Immunology and Microbiology courses and collaborates with local veterinarians. The P.D. has submitted parts of this work for publication during the project period and will continue efforts to publish all research pertaining to thisproject. What do you plan to do during the next reporting period to accomplish the goals?During the next project period we will: 1)implementAim 3 challenge studies in collaboration with Dr. McGuire (University of Idaho), 2) begin analysis of newly identifiedS. aureus antigens for incorporation into a mucosal mastitis vaccine, 3) complete data analysis of trial results, 4) express and purify Streptococcus uberis CTA2/B chimeric vaccinesand 5)compileand submit results in multiple manuscripts.During this period we will also continue to workwith the Boise State Office of Technology Transfer to obtain industry partners and identify new collaborations toadvance these efforts to make an effective vaccine to prevent bovine mastitis.
Impacts
What was accomplished under these goals?
Aim 1) during this project period we designed and implemented a second immunogenicity trial to assess responses to a mucosal bovine vaccine to prevent Staphylococcus aureus mastitis. This trial was distinct from the first, performed in 2015, in that: a) the sample size was increased from 7 per group to 11 cows per group, b) the concentration of total antigen delivered was increased from 900ug to 1200ug in a total of two doses instead of three doses, and c) more samplings of blood were taken to assess immune responses and to collect cells for flow cytometry. The vaccine was delivered intranasally in this trial, as in Trial 1. Anti-IsdA and ClfA ELISA data from Trial 2 is supportive of Trial 1, but indicates that a three-dose schedule, even with lower vaccine concentrations, may be optimal for immune responses uponfreshening. Cellular responses from Trial 2 indicated a mixed Th2-Th1 reponses with trend to Th2. The combination of Trial 1 and Trial 2 data are currently being incorporatedinto in a manuscript to be submitted early in 2017. Aim 2) we have assayed a collection of over 40 strains of bovine Staphylocccus for the presence and conservation of the IsdA antigen, as well as a number of other extracelular matrix binding adhesins. Results indicate that isdA is conserved, but sequencing indicates there are at least two common variants of this protein. This work is incorporated into a short manuscript for submission in November 2016. In addition, we have determined that IsdA is expressed in the milk of infected animals and also in vitro in milk, and this leads to increased binding to fibronectin. This work was submitted in 2016, but reviewers indicated that construction and characterization of an isdA mutant would enhance efforts. Also, within this aim, we have made substaintial progress toward the identification of new surface antigens from bovine S. aureus that are immunogenic in mastitic cows. This work has combined 2D gel electrophoresis, immunoblotting, mass spectrometry and proteomics. Thus far we have identified 4-5 S. aureus surface adhesins and/or virulence factors that represent newantigens to be characterized for a bovine vaccine. Future work on this aim will involve in silco analysis, cloning, purification and immune analysis of these antigens. Lastly, we have constructed clones for the expression of cholera toxin CTA2/B chimeras containing the Streptococcus uberis antigens SUAM and mtuA. Purification of these vaccines will likely occur within the next project period. Aim 3)An IACUC protocol has been constructed for challenge studies, and is currently in progress through the Boise State and Univeristy of Idaho committees. IBC review has been approved for these studies. This final trial (Trial 3) will utilize a total of 9 cows and will assess the ability of three intranasal doses (total of 1200 ug) of IsdA-CTA2/B and ClfA-CTA2/B to protect cows from challenge with isogenic S. aureus mastitis. The Trial will take a total of 60 days and will occur in the spring and early summer of 2017 in collaboration with Dr. Mark McGuire of the University of Idaho.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Misra, N., Wines, T.F., Knopp, C.L., McGuire, M.A., J.K. Tinker (2016) The low iron conditions of milk promote Staphylococcus aureus isdA expression and fibronectin binding. Veterinary Research (submitted Aug 2016, rejected Oct 2016)
- Type:
Journal Articles
Status:
Other
Year Published:
2016
Citation:
Misra, N., Wines, T.F., Knopp, C.L., McGuire, M.A., and J.K. Tinker (2016) Variants of the iron-regulated surface protein A from human and bovine Staphylococcus aureus. FEMS Micro Letters (to be submitted Nov 2016).
- Type:
Other
Status:
Accepted
Year Published:
2016
Citation:
Knopp, C.J (Dec 2015). The effect of a novel vaccine on Staphylococcus aureus binding and colony formation. M.A. Project, Biological Sciences, Boise State University.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Weaver, H.J., Wines, T.F. and J.K. Tinker. Assessment of antigen specific humoral immune responses in dairy cows after administration of IsdA-CTA2/B and ClfA-CTA2/B vaccine. ASM Intermountain Branch Annual Conference, Salt Lake City, UT, April 2016. Winner, best undergraduate poster $100.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Chapman, K., Weaver, H., and J.K. Tinker. Purification of Staphylococcus aureus IsdA and ClfA antigens to promote bovine vaccine development. ASM Intermountain Branch Annual Conference, Salt Lake City, UT. April 2016.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Brown, K. and J.K. Tinker. Evaluation of anti-IsdA antibodies to block Staphylococcus aureus adhesion in vitro and construction of new vectors for IsdA chimera vaccine expression. ASM Intermountain Branch Annual Conference, Salt Lake City, UT. April 2016.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Misra, N., Wines, T.F. and J.K. Tinker. A mucosal vaccine for bovine mastitis using cholera toxin A2/B adjuvant. International Society for Vaccines Annual Conference, Boston, MA. Oct 2016. Winner, student travel award $500.
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Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Efforts during the project period were research based and focused on completinga pilot Staphylococus aureus vaccine trial using a large local commercial dairy farm and performing genomic and transcriptional analysis of bovine S. aureus.Thus, specifically during this project period the target audience of the dairy farmer was reached. In addition, collaboration with local veterinarians has produced contactsduring this project period within the vaccine industry for translation of this research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student training: Neha Misra, Ph.D. candidate, is supported as a Research Assistant on this project for two years. Neha has been able to attend one regional conference (NIH IDea Western Regional Conference) and onelocal conference(Idaho Academy of Sciences). Colton Knopp, M.A. candidate, began work on this project in the fall of 2014 and will complete his studies in Decof 2015. Onerotation Ph.D. student; Eric Sweicki, was alsotrained on experimentation specific to this project. Undergraduate training: Laura Rogers, Hannah Weaver, Connor Richmond,Elizabeth Stewart, andDana Claryare Boise State Undergraduates who have performed research on thison this project. In addition a number of volunteer undergraduates, including: Kimmy Brown and Montana Markshave been trained on project related techniques and experimentation. Travel to the NIH IDea Western regionalconference for Neha Misra and the PI was supported during this project period. How have the results been disseminated to communities of interest?The P.I. has desseminated research efforts to the media in the form of a newspaper article on technology transfer (Idaho Statesman, published 12-29-15), and to industry in the form of a presentation to the Idaho Technology Council Capital Conference (Oct 2015). The PI has disseminated this work to the science community through presentations at scientific conferences (Keystone Conference on Veterinary Immunology, Jan2015, Idaho Academy of Sciences, March 2015, Idaho Conference on Undergraduate Research, July 2015 and the NIH Idea Western Regional Conference, Oct 2015) as well as efforts on submission and preparation of manuscripts.The PI continuesto workwith the Central District Health Immunization Advisory Committee to desseminate public awareness and vaccine education and will teach a course on Vaccinology in the spring of 2016. What do you plan to do during the next reporting period to accomplish the goals?During the next project period efforts will be made to: 1) complete the bovine immunogenicity Trial #2 (Objective 1), 2) begin vaccine challenge trial (Objective 3), 4) complete 2D gel electrophoresis to identify novel S. aureus antigens (Objective 2), 5) submit manuscript on transcription of S. aureus adhesins in milk, 5) submit manuscript on IsdA immunogenicity study, 6) purify and characterize S. uberis cholera toxin chimeras (Objective 2), 6) compile research fora review article.
Impacts
What was accomplished under these goals?
Aim 1) during this project period we completed assays on all bovine samples fromTrial #1 bovine vaccineimmunogenicity study. Trial 1began 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 has shown a significant increase in anti-IsdA and anti-ClfA responses in milk from vaccinated cows and a trend of increased anti-IsdA and anti-ClfA responses in serum. No cows from any group became infected with S.aureus during the study and results indicate no signicant effect on SCC and positiveoverall safety.Current efforts are focused on protocols for cellular analysis andimplementing Trial #2 with a higher dose and more animals per group.Aim 2) during this project period we have continued to developprotocols for 2D gel electrophoresis to identify novel S. aureus vaccine candidates. An NIH pilot COBRE grant was received to perform 2D gel electrophorsis on proteins that specifically bind to extracellular matrix molecules. These studies are currently underway. In addition we have completed studies to characterize ECM binding of bovine S. aureus grown in milk and the ability to block ECM binding with serum from vaccinated cows. Two Streptococcus uberis vaccineantigens have been cloned into CTA2/B expression vectors, however, thus far we have been unable to purify properlyfolded S. uberischimeras.Lastly, a publication was submitted that characterized the expression andvariablity of IsdA from bovine milk, and a second manuscript will be submitted soon on the expression of ECM adhesins from S.aureus grown in milk.Aim 3) during this project period we have continued collaborative discussions and IACUC protocol development for challenge studies at the University of Idaho. Efforts on this aim may be modified during the next project period depending on results from immunogenicity trials.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Wines, T.F., Misra, N., Williams, J.E., McGuire, M.A. and J.K. Tinker (2015) Characterization of Staphylococcus aureus IsdA from Bovine Isolates. Veterinary Microbiology. September 2015
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Staphylococcus aureus IsdA and ClfA - cholera toxin A2/B fusions as mucosal mastitis vaccines. Wines, T.F, Misra,N. and J.K. Tinker. Keystone Symposia on Immunity to Veterinary Pathogens: Informing Vaccine Development. Jan 20-25, 2015. Keystone,CO
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Weaver, H.J., Wines, T.F. and J.K. Tinker (2015) Assessment of antigen specific humoral immune responses in dairy cows after administration of IsdA-CTA2/B + ClfA-CTA2/B vaccine. Idaho Conference of Undergraduate Research. Boise, ID July 28, 2015
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Richmond, C.G, Wines, T.F and J.K. Tinker (2015) Staphylococcus aureus ClfA-cholera toxin A2/B as a primary vaccine candidate for bovine mastitis. Idaho Conference of Undergraduate Research. Boise, ID. July 28, 2015
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Stewart, E, Wines, T.F. and J.K. Tinker (2015). Cloning, protein extraction and purification of antigens for a dairy cow vaccine. Idaho Conference for Undergraduate Research. Boise, ID, July 28, 2015
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Misra, N. and J.K. Tinker. (2015) Regulation of Staphylococcus aureus adhesin gene expression in vitro simulating bovine mastitic conditions. 2015 NIH IDea Western Regional Conference. Coeur d'alene, ID, Oct 3, 2015.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Tinker, J.K. (2015) Mucosal vaccines to prevent Staphylococcus aureus in cows. Idaho Technology Capital Conference. Boise ID. Oct 21, 2015
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Progress 01/01/14 to 12/31/14
Outputs
Target Audience: Efforts during the project period focused on designing and implementing a pilot Staphylococus aureus vaccine trial using a large local commercial dairy farm. Thus, specifically during this project period the target audience of the dairy farmer was reached. Changes/Problems: No major changes occurred during this project period and immunogenicity trials (Objective 1) were performed as proposed with no safety concerns. However, major changes will occur during the next project period due to PI, co-PI and consultant concerns of the novel route of vaccine delivery proposed (intramammary and intravaginal). It was originally proposed that immunogenicity would be assessed in a commercial dairy herd using these routes (Trials 2 and 3), however, it has been deemed necessary to ensure the safety of these routes prior to a large study in a commercial herd. Thus IACUC protocols will be developed in collaboration with Dr. Mark McGuire (Co-PI) to perform a small pilot prior to initiation of the proposed Trial #2/3. In addition, protein preparation has been found to be significantly more time consuming and expensive then proposed. Thus Trials 2 and 3 will be combined to reduce the number of animals and amount of vaccine required to assess the immunogenicity using these routes of delivery. IACUC approval has been obtained for this modification. What opportunities for training and professional development has the project provided? Graduate student training: Neha Misra, Ph.D. candidate, is supported as a Research Assistant on this project for two years. Neha has been able to attend one regional conference (ASM Intermountain Branch) and two local conferences (Boise State Graduate Research and the Idaho Academy of Sciences). Colton Knopp, M.A. candidate, began work on this project in the fall of 2014 and will complete his studies in the fall of 2015. Two rotation Ph.D. students, Sheenah Bryant and Nathan Redman also were trained on experimentation specific to this project. Post-doctoral training: Farzana Siddique, Ph.D. Assistant Professor, Arid Agricultural University, Rawalpindi, Pakastan, worked in the lab from May 2014 to November 2014. Farzana constructed and characterized the S. uberis cholera toxin A2/B chimeras. Undergraduate training: Emily Price and Laura Rogers are two Boise State Undergraduates who have been supported part-time on this project. In addition a number of volunteer undergraduates, including: Kimmy Brown, Connor RIchmond and Hannah Weaver have been trained on project related techniques and experimentation. Travel to the ASM Intermountain conference for Neha Misra, Laura Rogers, Kimmy Brown and Emily Price was supported during the project period. How have the results been disseminated to communities of interest? The P.I. has desseminated the research efforts to the media in the form of a newspaper article on technology transfer (Idaho Statesman, not yet published) and an article in the Boise State Research magazine (Explore, published Feb 2015) during the project period. In addition, an invited lecture was given in Sept 2014 in the Department of Pharmacy, Idaho State University, Meridian, ID as well as an invited talk to Meridian Medical Arts High School, Meridian ID (Nov 2014) and the Idaho Conference on Undergraduate Research, Boise, ID (July 2014). The PI continued to work during the project period with the Central District Health Immunization Advisory Committee to desseminate public awareness and vaccine education. What do you plan to do during the next reporting period to accomplish the goals? During the next project period efforts will be made to: 1) complete the bovine immunogenicity Trial #2 (Objective 1), 2) complete IACUC protocols to begin vaccine challenge trial (Objective 3), 3) improve techniques and obtain instrumentation to increase protein production (Objectives 1 and 3), 3) complete 2D gel electrophoresis to identify novel S. aureus antigens (Objective 2), 4) complete qPCR studies and submit manuscript on this work (Objective 2), 5) submit manuscript on IsdA immunogenicity study, 6) purify and characterize S. uberis cholera toxin chimeras (Objective 2), 6) begin cholera toxin adjuvant review article.
Impacts
What was accomplished under these goals?
Aim 1) during this project period we developed protocols for the proposed bovine immunogenicity trial #1. These protocols were approved by the Boise State IACUC, the Idaho State Veterinarian and the USDA Center for Veterinary Biologics. Trial #1 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. Prior to the start, all study cows were screened for low anti-isdA responses, no evidence of S. aureus, and no evidence of BLV infection. Preliminary data analysis suggests there is a trend of increased anti-IsdA responses in vaccinated cow serum and milk and reduced presumptive staphylococcal species in milk in vaccinated cows. Current efforts are focused on designing and implementing Trial #2 and compiling all data from Trial #1. Aim 2) during this project period we have developed protocols for 2D gel electrophoresis and have completed one experiment that has identified two conserved S. aureus cytosolic proteins as highly immunogenic in infected bovine milk samples. These studies are currently being repeated using different protein isolation methods with the goal of identifying surfaceS. aureus vaccine candidates for further study. In addition, we have refined quantitiative PCR protocols to analyze virulence factor expression under low iron conditions similar those found in bovine milk. A number of potential vaccine candidates have been evaluated by qPCR so far, including, isdA, isdB, clfA, fnbpA, fur, nuc, and hla. We have characterize expression in human and bovine S. aureus strains as well as S. hemolyticus. This work will be compiled for publication within 2015. Lastly, we have identified and cloned three virulence factors in Streptococus uberis including: mtuA, pauA, and suaM, and have constructed vectors for cholera toxin A2/B fusions to these antigens for incorporation into a potential multi-species vaccine. Aim 3) during this project period we have continued collaborative discussions and IACUC protocol development for challenge studies at the University of Idaho. Efforts on this aim may be modified during the next project period depending on results from immunogenicity trials.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Staphylococcus aureus IsdA and ClfA - cholera toxin A2/B fusions as mucosal mastitis vaccines. Wines, T.F, Misra,N. and J.K. Tinker. Keystone Symposia on Immunity to Veterinary Pathogens: Informing Vaccine Development. Jan 20-25, 2015. Keystone, CO. Abstract accepted November 7, 2014.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Characterization of IsdA from Staphylococcus aureus as a promising vaccine antigen to prevent bovine mastitis. Misra, N., Wines, T., Jeffries, S. and J.K. Tinker. American Society for Microbiology Intermountain Branch Annual Meeting. March 7-9, 2014. Provo, UT.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Purification of cholera toxin - IsdA, IsdB, ClfA and SrdE fusions for use as a potential Staphylococcus aureus vaccine. Rogers, L. and J.K. Tinker. American Society for Microbiology Intermountain Branch Annual Meeting. March 7-9, 2014. Provo, UT.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Construction and Characterization of Non-Toxic Bacterial Enterotoxins as Vaccine Adjuvants. Lavanya Vempati Master of Science, Biology 2014.
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