Performing Department
(N/A)
Non Technical Summary
Mastitis, or inflammation of the udder, is considered the most economically significant disease affecting dairy cattle worldwide; costing the industry an estimated $2 billion a year. The bacterium Staphylococcus aureus is a main cause of mastitis that can establish subclinical infection, and result in long term reductions in milk yield. Our innovation is a subunit vaccine that can be administered to mucosal and cutaneous surfaces to induce S. aureus-specific immunity. This vaccine uses a platform based on the enterotoxin, cholera toxin (CT). CT is a well-established immunostimulatory adjuvant that induces local and systemic immunity to block colonization and prevent disease. A2/B fusions, or chimeras, of CT (CTA2/B) are non-toxic, easily purified, and safe and immunogenic after intranasal delivery in cows. Our long term goal is to produce a cost-effective vaccine to eliminate S. aureus mastitis in dairy cows. We hypothesize that the CTA2/B vaccine platform can be adapted to incorporate additional targeted S. aureus antigens, and that this platform can be delivered safely to the bovine udder to induce specific immunity. For this Phase I STTR we propose feasibility studies to: 1) expand and enhance vaccine protective capacity by incorporating targeted S. aureus antigens, and 2) assess vaccine safety, immunogenicity and efficacy after intramammary vaccination in cows. Studies will support technology transfer to Pentamer Biologics, LLC; a small business dedicated to vaccine development in collaboration with Boise State University. Studies also support the mission of USDA NIFA, to advance agricultural research, and the priority area of Animal Production and Protection.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
(N/A)
Goals / Objectives
Our long term goal is to produce a cost-effective vaccine to eliminate S. aureus mastitis in dairy cows. We hypothesize that the CTA2/B vaccine platform can be adapted to incorporate additional targeted S. aureus antigens, and that this platform canbe delivered safely to to the bovine udder to induce specific immuntiy. To test our hypothesis and support the long-term goal of developing a vaccine to reduce or eliminate S. aureus mastitis in dairy cows, we propose the following two technical objectives:Objective 1: Construct a rationally designed multivalent vaccine to prevent clinical and subclinical S. aureus mastitis by incorporating additional targeted antigens. A need remains to expand vaccine protection by incorporating additional antigens from multiple virulence pathways. To address this need, we will build upon previous omics studies to prioritize candidates and expand the current CTA2/B vaccine. We will score a pool of antigens in silico based upon: 1) vaccine antigen prediction, 2) immunoinformatics and epitope prediction, and 3) genomic antigen conservation. Up to ten final candidates will be cloned for chimera expression, using original and newly constructed CTA2/B vectors. Resulting chimeras will be purified, assessed for stability and binding and combined into a new multivalent vaccine.Objective 2: Determine vaccine safety and efficacy after intramammary vaccination in a pilot bovine challenge trial. Intramammary vaccination has been successful in bovine trials, however the safety and efficacy of this route using enterotoxin-based adjuvants is unknown. To address this question, we will perform a randomized/blinded challenge trial using six cows vaccinated and boosted through the intramammary route during milking. Milk and serum will be collected for cytokine, antigen-specific antibody and T-cell proliferation assay. Clinical outcomes and bacterial shedding will be used to assess vaccine efficacy.
Project Methods
Objective 1 Efforts: Construct a rationally designed multi-valent vaccine to prevent clinical and subclinical S. aureus mastitis by incorporating targeted antigens. We will build upon our previous efforts to identify S. aureus candidate vaccines and select 10 priority antigen candidates using in silico methods. Selected candidates will be cloned into vectors for CTA2/B fusion expression. These constructs will then be purified and assessed for predicted protein composition and receptor-binding. In silico vaccinology. We have designed an in silico workflow for antigen selection. Efforts are expected to occur within the first half of the funding period. We will score the current 200 candidates based on: 1) vaccine antigen prediction, 2) immunoinformatics and epitope prediction, and 3) genomic antigen conservation. Vaccine antigen prediction. Vaxign rapidly assesses proteins for subcellular localization, transmembrane helices, adhesion probability and homology to host proteins.Antigens with an ML score of 90 or above and no homology to human/mouse/pig proteins will be selected for further study. Immunoinformatics. There are several free computational T and B cell epitope prediction tools, and Vaxign (Vaxitop) includes B and T cell epitope prediction. We will use Vaxitop to identify human supertype MHC I and I epitopes at a p value of ≤ 0.005. Epitopes will be compared to those identified using the Immune Epitope Database and Analysis Resource (IEDB) and NETBoLAIIpan. We will select antigens that have at least one B cell and one T cell epitope located with a conserved region.Candidate antigens will be prioritized as those with a balance of B cell and Th1/Th2/Th17 T cell epitopes. Genomic antigen conservation. The annotated sequences of bovine RF122, LGA251, and Newbould 305 are available in NCBI to assess sequence conservation of prioritized antigens. We will screen the top 20 candidates for sequence identity using tBLASTn and the 27 S. aureus sequences within BioProject PRJNA609123Candidates will be narrowed to the top 10 with an overall average identity of 90% or above.Production of CTA2/B chimeras. Cloning and purification. The top 10 priority candidates will be isolated by PCR and directionally cloned in the pARLDR19 vector for CTA2/B chimera expression. Expression of the antigen-CTA2 fusion and CTB will be stimulated by arabinose and holotoxin-like proteins will be isolated from the E. coli periplasmic space using D-galactose affinity chromatography.Confirmation of CTA2/B folding will be performed by SDS-PAGE and anti-CT western blot. Analysis of CTA2/B stability and binding. Ganglioside GM1 binding ELISA assays using anti-CT antibodies will be used to assess chimera receptor-binding capacity. Alternative CTA2/B expression vectors. Two additional CTA2/B expression vectors have been constructed based upon the pARLDR19 vector. Data interpretation, analysis and evaluation. A protocol for systematic antigen and epitope proritization will be developed.Vaccine composition for Objective 2 will be decided by which of the priority antigens or epitopes can be most stably produced and purified as a CTA2/B chimera. Evaluation of methods will occur in Objective 2, by the determination of antigen-specific immune responses to individual antigens within the resulting mult-valent vaccine.Objective 2 Efforts: Determine vaccine safety and efficacy after intramammary vaccination in a pilot bovine challenge trial. We will complete a short vaccination and challenge trial using a homologous isolate of S. aureus to assess safety, antigen-specific humoral and cellular immune responses, and efficacy to reduce bacterial shedding. Results will provide essential evidence of the feasibility of this CTA2/B platform for intramammary vaccination of cows. Bovine immunization and challenge. Vaccine composition. The vaccine will consist of purified protein suspended in saline buffer. Location and animals. The experimental trial will occur at Johnson Research LLC in Parma, Idaho, Six healthy Holstein cows in lactation number 1-3, and approximately the same number of days in milk, will be chosen. Chosen animals will be removed from the herd for the trial period (33-35 days). Milk will be discarded during trial period. Immunization. Animals will be randomized into two groups and all members of the team, except for the P.D., will be blinded. Three cows will be immunized into all four quarters with 2.5 mL of 100 mg (400 mg/cow) of purified S. aureus antigen-CTA2/B vaccine (Group 1), and three cows will receive an equal volume of 1x PBS + glycerol vaccine vehicle control (Group 2). The project supervisor and/or technician will visually inspect cows daily after vaccination for signs of inflammation and mastitis as well as breathing difficulties, weight loss, infection, lethargy, or other abnormal conditions that would warrant euthanasia. On Day 14, animals will receive a booster vaccination of equivalent concentration and volume. Experimental mastitis. On Day 21, seven days after booster, all animals will be challenged intramammary into two opposite quarters with 1 mL of 200 CFU/mL (400 CFU/cow) of the homologous vaccine S. aureus strain (Newbould 305). Challenge will occur aseptically. On Day 21, after milking, 1 mL of the S. aureus suspension will be inoculated into two quarters of each cow after each teat is disinfectedTwo quarters will be left uninoculated. Clinical outcomes, sampling and data collection. Clinical assessment. Cows will be monitored immediately after challenge and daily during challenge period. Clinical outcomes will be scored and documented by the attending veterinarian, and will include those for temperature, general appetite, soreness and swelling of the teat, and appearance of milk, including the presence of clots. Sample collection and schedule. Serum will be collected nine times during the trial (days -2, 1, 10, 14, 17, 21, 24, 27, 31) for immunological analysis. Whole blood will also be collected once during the study to isolate PBMC, as described below (Day 21). Milk will be collected for SCC determination and S. aureus culture on days -2, 1, 10 and 14 prior to challenge and then every day during challenge (Days 21 - 31). After collection, milk and blood will be packaged and shipped as described below. Bacterial and immunological analysis.Pooled quarter milk will be fixed for SCC/nutritional content. Quantification of intracellular S. aureus will occur using fresh milk incubated in gentamicin and plated for CFU. Milk will also be processed and stored for antibody purification and ELISA using purified IsdA and ClfA.Whole blood will be used to isolate PBMC on Day 21 after vaccination and before challenge. PBMC will be grown and stimulated with antigens for assessment of cellular proliferation using flow cytometry. Antigen-specific opsonophagocytic assays (OPA) can also be performed using antibodies purified from milk or serum to determine if they are functional in vitro, as described (Misra et al., 2017). Data analysis, interpretation and evaluation. Data will include safety assessment, antigen-specific immune (B and T cell) responses, and colony-forming unit (CFU) assessment of vaccine efficacy.Positive outcomes that will support a Phase II application and continued vaccine development will include: 1) significant antigen-specific antibody production in the milk and blood over control, 2) significant antigen-specific T cell response over control and 3) significant reductoin in milk bacterial shedding over control by the end of the 10 day challenge period. Lastly, the vaccine must show a positive safety profileto move forward including: 1) lack of evidence of severe mammary gland or systemic inflammation in vaccinated animals and 2) lack of evidence of clinical mastitis, over that induced in controls, after challenge. The clincal vaccine safety profile will be further refined in collaboration with the project veterinarian.