Performing Department
Vaccines
Non Technical Summary
Salmonella is a bacteriathat causessevere stomach and gut distressinpeople andis the leading cause of food-borne illnesses resulting in hospitalization and death in the US. The resulting economic losses caused bythisbacteriain the livestock industry are staggering with much of the loss attributed to recalls, permanent disruption of the gut lining impairing nutrient absorption, and necessary culling of sickanimals. Furthermore, antibiotic resistance has now made routine antibiotic use in livestock unsustainable. A significant problem with this bacteriais that it cancolonize the lymph nodes of beef animals. Lymph node removal at processing plants is not financially realistic. Prevention of infection and colonization in beef cattle and other speciesis the optimal solution. Current vaccines don't provide protection againstmost versions of thisSalmonella bacteria. We have developed a subunit vaccine that if proven successful, will provide broad protection against this bacterial infection. Proof of concept in mice is completed.This study is designed todemonstrate vaccination protectionfrom this bacteria incalves. Samples will be collected to assess vaccine immune responses and to understand if it confers immunity in the animal, thus preventing exposure of the bacteria to people.Lymph nodes will be collected to assure that, in addition to reducing the bacteria shed form the animal's feces, the infectionwill not show up in the lymph nodes, and this means in won't show up in hamburger or other beef products. When successful, this project will move our technology closer to commercialization, including a two dose vaccine,to lower or eliminate the Salmonella burden in beef, swine and poultry to protect America's food security as well as our population from this devastating pathogen.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Goals / Objectives
The primary goal of this project is to demonstrate thatour T3SAsubunit vaccine results in non-shedding of Salmonella challenged animals (based on a previously established and recognized challenge model) with no lymph node carriage. Success of the homologous vaccine will then trigger a heterologous challenge of the animal model (and target species). A viable product that meets producers' needs to help control and eliminate Salmonella infections with a cost-effective, broadly protective vaccine will improve beef production and lessen the risk of contamination for both pet products and human foodproducts.Objectives:1) Complete protein purification of S1F and S2F and vaccine preparation by April 1 2016.2) Obtain and acclimatize certified, Salmonella-free Holstein calves at the Veterinary and Biomedical Research Center (VBRC), Manhattan, KS by April 30, 2016.3) Follow vaccination schedule, sample collection, challenge, and results collectionas per protocol and within budget and collate results for analysis by July 31, 2016.4) Preparestudy report, recommendations for further studies and full submission of final REEportbyDec 31, 2016.
Project Methods
The recombinant proteins, S1F and S2F, will be madeby Dr. Wendy Picking at the Kansas Vaccine Institute (KVI) at the University of Kansas. Briefly, for S1F, the sipD-sipB gene fusion has been cloned into pET9d which is kanamycin resistant and the gene for the cognate SipB chaperone SicA, is cloned into pACYC which is chloramphenicol resistant. The plasmids are co-transformed into E. coli Tuner(DE3) for expression. The bacteria are grown to induce protein expression. After3 hours, bacteria are collected by centrifugation, lysed, and inclusion bodies collected again in the pellet, which is resuspended, and then clarified by centrifugation and loaded onto a nickel charged IMAC column. The column is washed and the protein eluted. The eluted protein is passed over a Q column with the eluted protein slowly refolded by step dialysis. The solution is passed again over an IMAC column to remove the chaperone which was released from the complex by the detergent. Fractions are collected and the final dialysis step exchanges the proteins into phosphate buffered saline (PBS) containing LDAO which is required to maintain solubility of the proteins and has a GRAS (Generally Regarded As Safe) Profile.The proteins will be concentrated to 5 mg/ml and stored at -80 °C. The adjuvant will be monophosphoryl lipid A (MPL; Sigma Chemical Co.) combined with dipalmitoylphosphatidyl choline (DPPC) in a 4:1 molar ratio. Two milligrams of each protein will be combined with ~ 2 mg of Alhydrogel (AH) and allowed to bind for one hour at room temperature. Currently, binding isotherms are being performed to determine the actual optimal Alhydrogel ratio per unit protein. MPL (100 μg) will be added to the formulation with a final vaccine formulation containing 1 ml. A live attenuated ΔSPI-1/2 S. Typhimurium strain will be used for the positive control and will be grown overnight at 37 °C with shaking, collected by centrifugation, washed and finally resuspended in PBS at 1x107cfu/ml. The negative control will be PBS only.Calf experiment will be performed at the Veterinary and Biomedical Research Center (VBRC), Manhattan, KS by Kelly Lechtenberg, DVM, PhD.who has established himself and his team as a leader in veterinary large animal contract research. All animals use will be approved by the VBRC Institutional Animal Care and Use Committee. Holstein steers, 200 pounds, will be obtained from one of the commercial Holstein calf vendors that can assure study candidates are Salmonella-free and have not been given any commercial vaccine against Salmonella by pre-screening fecal samples and by control of vaccination and management protocols. Fecal samples will be obtained prior to the experiment to ensure no Salmonella is detectable. Calves will be housed individually in indoor pens. Calves will initially be fed twice daily (bottle or pail) with a commercial milk replacer appropriate for the age of the calves. As calves start to consume non-medicated dry fed, milk feeding will be reduced to once daily to enhance dry feed consumption. Calves will have access to fresh water ad libitum.Two groups (n=10/group) of calves will be vaccinated subcutaneously three times on days 0, 14, and 28, with either PBS or the S1F+S2F (2 mg each) adsorbed to AH then admixed with 100 μg MPL (see above vaccine formulation). Our goal is to get the end product down to two doses. The ΔSPI-1/2 strain (2 ml) will be fed to another group of 10 calves on days 0 and 28 by mixing with milk. Groups of ten were chosen as this is the minimum number that is required to obtain significant results in both the challenge experiment and the immunology determinations. In addition, power analysis performed in our group suggests that a number of 10 animals per group are enough to discern a difference of ~20% in protective capacity.Sample collection. Blood will be drawn at 0, 14, 28, and 42 to obtain serum (from clotted blood) and peripheral blood mononuclear cells (PBMCs, from Heparinized blood) via standard procedures. Fecal samples and saliva will be collected at these times as well. One mg of fecal contents will be suspended in 1 ml of PBS containing sodium azide and the suspension shaken vigorously by vortexer for 15 min. The suspension is clarified and the supernatant will be frozen at -80°C until analyzed by ELISA. The saliva is transferred via centrifugation into a 50 ml conical tube from the applicators that are in a 15 ml conical tube with a hole in the bottom. Kinetics of serum IgG and fecal IgA titers will be determined. The Immunological Core of the KVI will perform the ELISAs to determine the antigen-specific IgG and IgA levels. Dr. Martinez-Becerra has extensive experience with isolating the appropriate fraction from the blood, fecal samples and saliva and performing ELISAs. While not published, the preliminary data provided for the protective efficacy of SseB in calves included determination of the kinetics of serum IgG and fecal IgA production by the calves. PBMCs separated from blood samples will be incubated in plates with SipD, SipB, SseB, and SseC to assess the frequency of antibody secreting cells. Similarly, after stimulation with the antigens, the cells and supernatant will be assessed for IFNγ and IL-17 levels via flow cytometry and ELISA respectively. Dr. Martinez-Becerra also has experience with these assays.Statistical analysis will be performed using Graphpad prism and FlowJo softwares. Differences among treatments will be analyzed using ANOVAs and t-tests. A P value < 0.05 will be considered significant for all analysis.Go/No Go to Challenge. Based on previous immunological results, the titers for the anti-SipD, SipB, SseB, SseC-specific IgG need to be at least 103-104 ELISA units/ml with IgA units being 103 ELISA units/ml. Background is less than 25 EU/ml. Similarly, there should be 100 IgG and 50 IgA antibody secreting cells (ASC) specifically directed against each antigen per 106 peripheral blood mononuclear cells (PBMCs). In a previous calf experiment (see challenge results below), we have seen an increase in IFNγ and IL-17 levels and while these are expected, they will not be used to Go/No-Go decisions. If these conditions are not met, another vaccination will be performed. We are confident that three vaccination will stimulate a complete immune response since maximal IgG and IgA titers were detected at the sampling associated with day 48 vaccination previously, although our goal is to get to 2 dose vaccination for commercial reasons.S. Typhimurium challenge. We will challenge the calves orally with S. Typhimurium SL1344 StrRwhich is the inoculum required to result in Salmonella-positive peripheral lymph nodes. A sample of the final inoculum will be subjected to dilution plating to determine the actual final inoculum (prior to challenge, growth, manipulation and dilution will be performed until the dilution plating is within 1% error on 3 consecutive trials). Upon challenge, weight loss will be monitored over a seven day period (Day 0& Day 7). Temperature will be monitoredand health scores will be determined in the morning (daily). Fecal shedding will be monitored over a seven day period by resuspending 1 mg of fecal matter in 10 ml of PBS by vortexing, and serial dilutions plated onto TSB+streptomycin (50μg/ml). At the end of the study, the calves will be euthanized, necropsies performed, and the Salmonella burden assessed in the matter of the gastrointestinal tract and in the major lymph nodes.