Progress 01/01/24 to 12/31/24
Outputs
Target Audience:The results of the project were presented at the 2024 Annual PSA meeting to the audience group that consists of professors, staff, graduate students from academia, and scientists and personnel from poultry or related industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We trained 3 Ph.D. students through this project. They have acquired new knowledge and skills to understand andperform the experiments in this project. How have the results been disseminated to communities of interest?We presented the results from this project through presentations at the annual Poultry Science Association meeting. We presented 4abstracts through oral presentations (shown below). Abigeal I. Omolewu, Christine N Vuong, Santiago U. Diaz, Adil S. Al-Ogaili, Seong Wook Kang, B. M. Hargis, and Young Min Kwon. Development and Evaluation of DNA Aptamer Rolling Circle Amplification (RCA) Platform as a Vaccine Complex to Control Salmonella in Broiler Chickens. July 15-18, 2024. PSA Annual Meeting, Lousville, KY (Oral presentation) Santiago Uribe-Diaz, Abigael I. Omolewu, Jossie M. Santamaria, Chrysta N. Beck, Christine N. Vuong, Guillermo Tellez-Isaias, Gisela F. Erf, Billy M. Hargis, Young Min Kwon.Evaluation of the cellular immune response initiated by DNA aptamer-based experimental Salmonella vaccine complex using the growing feather pulp cutaneous test in broiler chickens.July 15-18, 2024. PSA Annual Meeting, Lousville, KY (Oral presentation) Deborah Olubanjo, Hanan Alkabkabi, Seong Wook Kangand Young Min Kwon.Genetic determinants of Salmonella Typhimurium required for growth in chicken cecal extract media.July 15-18, 2024. PSA Annual Meeting, Lousville, KY (Oral presentation) Chapagain Arjun, Deborah Olubanjo, Asim Abbasi, Seong Wook Kang, and Young Min Kwon.Genetic transformation of chicken gut commensal bacteria using random transposon mutagenesis.July 15-18, 2024. PSA Annual Meeting, Lousville, KY (Posterpresentation) What do you plan to do during the next reporting period to accomplish the goals?Encouraged by the efficacy of our RCA vaccine complex in eliciting immune responses and reducing gut colonization by Salmonella, we will perform vaccination of breeder hens to determine if the antibodies produced by RCA vaccine can be carried to the offsprings in the form of the maternal antibodies. We will continue to develop improved DNA complex design with multiple advantages: 1) simpler and cheaper to produce, 2) equal or higher vaccination efficacy, and 3) increased versatility to use various types of antigens.
Impacts
What was accomplished under these goals?
We performed the chicken challenge study using S. Enteritidis strain to evaluatethe two designs of the CD40-targeting RCA vaccine complex for the immue response and reduction of gut colonization by Salmonella.The result showedthat RCA vaccine complexes (RCA-v3 and RCA-v5) significantly reduced S. Enteritidis colonization (0.98 log CFU/ml and 1.19 log CFU/ml, respectively, P ≤ 0.05) as compared to the challenged control (2.74 log CFU/ml) and killed S. Enteritidis group (2.76 log CFU/ml). Additionally, IgM antibody levelincreased significantly post-vaccination (P ≤ 0.05). We also performed aptamer-target docking simulations to better understand the interactions between the two CD40-targeting aptamers (SEQ3 and SEQ4) and chicken CD40 peptide. Our computer simulation result support our experimental data from HD11 cell activation assay. Additionally, we evaluated hybridization chain reaction (HCR) as a novel DNA scaffold to present CD40-targeting DNA aptamers. Wheneach aptamer(SEQ3 and SEQ4) was added individually (HCR-SEQ3 orHCR-SEQ4) or in combination (HCR-SEQ3-SEQ4), we found that only HCR containing SEQ3 (HCR-SEQ3 and HCR-SEQ3-SEQ4) showed significantly activation of HD11 cell, while HCR-SEQ4 showed no activation of HD11 cell(P ≤ 0.05). The result of this experiment suggests that only SEQ3 is involved in macrophage activation. This would allow simplification of our RCA complex design focusing only SEQ3 without losing any capacity activating HD11 cell.
Publications
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:The results of the study were presented at the 2024 Annual PSA meeting to the audience group that consists of professors, staff, graduate students from academia, and scientists and personnel from poultry or related industries. Changes/Problems:One of the goals of the project was to identify the genetic factors in Salmonella Typhimurium that are required for horizontal transmission of this pathogen within the broiler flock and to use the proteins or their peptides as the antigens in combination with the DNA vaccine complexes. However, we encountered both intrinsic and technical challenges that are difficult to overcome. We decided to omit this component from the project but added the new component (HCR-based DNA complex) to improve the vaccine effcacy. What opportunities for training and professional development has the project provided?We trained 3 Ph.D. students through this project. They have acquired new knowledge to understand the project and skills to perform the experiments in this project. How have the results been disseminated to communities of interest?We presented the results from this project through presentations at the annual Poultry Science Association meeting. We presented two abstracts through oral presentations (shown below). Abigeal I Omolewu, Christine N Vuong, Santiago U Diaz, Adil S Al-Ogaili, Seong W Kang, Billy M Hargis, and Young Min Kwon. Development and Evaluation of DNA aptamer rolling circle amplification (RCA) platform as a vaccine complex to control Salmonella in broiler chickens. July 15-18, 2024, PSA Annual Meeting, Louisville, KY. (Oral presentation) Santiago Uribe-Diaz, Abigeal I. Omolewu, Jossie M. Santamaria, Chrysta N. Beck, Christine N. Vuong, Guillermo Tellez-Isaias, Gisela F. Erf, Billy M. Hargis, Young Min Kwon. July 15-18, 2024, PSA Annual Meeting, Louisville, KY. (Oral presentation) In addition, we are currently working on 2 manuscripts to submit to peer-reviewed journals soon. What do you plan to do during the next reporting period to accomplish the goals? We will complete the evaluation of the two DNA vaccine complexes (versions 3 and 5) using a chicken challenge study. The results will be collected, analyzed, and summarized to prepare the presentations and manuscripts. We will complete the sample analysis from the growing feather model study and the pilot chicken challenge study to determine the humoral immune responses triggered by the DNA complex. We will complete the evaluation for the new DNA complex design based on HCR. If the result is similar to or better than the DNA complex designs based on RCA, we will also perform further evaluations using the growing feather model and chicken challenge study.
Impacts
What was accomplished under these goals?
Accomplishments The chicken challenge study was performed on a pilot scale to evaluate the two versions of the DNA complex (versions 3 and 5). The result showed that the group vaccinated with the version 3 DNA complex showed a significantly lower level of cecal colonization by the Major goals of the project Continued development of CD40-targeting DNA complexes for improved immune-enhancing capacity. Evaluation of the DNA vaccine complexes to control Salmonella through chicken challenge studies. What was accomplished under these goals? We established and have routinely used the macrophage activation assay using the HD-11 cell line. This assay gave reliable and consistent results on the level of macrophage activation through quantification of nitric oxide released from activated macrophage cells. We used this in vitro assay conveniently to screen and evaluate different design variations of CD40-targeting DNA complex. In addition to the previous versions of DNA complex, we evaluated new versions of DNA complex using HD-11 cell line assay and found that versions 3 and 5 produced the highest level of significant macrophage activation consistently. Therefore, these two versions of DNA complex (version 3 and 5) in combination with killed Salmonella cells were selected for further evaluation using 2 additional approaches: (1) evaluation of immune response using growing feather pulp cutaneous test model, and (2) chicken challenge model. The results from the growing feather model show that both the DNA complex (version 3) and commercial vaccine complex (mannosylated chitosan; positive control) induced heterophil and macrophage infiltration. However, only the DNA complex demonstrated a high and prolonged T- and B- lymphocyte infiltration during the recall immune response. challenge strain (S. Enteritidis) as compared to the group vaccinated with killed Salmonella cells. The main platform for producing DNA complex was the rolling circle amplification (RCA) product. In addition to RCA, we developed an alternative and potentially better platform using HCR (Hybridization Chain Reaction) to produce DNA complex, which may have certain advantages over the RCA-based platform. We will evaluate these two different platforms to compare the capacity to activate HD-11 cells.
Publications
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:We had great difficulty recruiting and getting the second graduate student (PhD) in place. There were residual problems which were partially COVID-related and associatied with acquisition of his visa. Santiago Uribe is now in place (Since August 14th) and will begin the animal work very soon. He is currently assisting with aptamer testing in vitro. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?One graduate student presentation was made at the Annual Meeting of the Poultry Science Association in Philidelphia this year, publication is in progress. We anticipate building momentum in the next year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major goals of the project 1. Evaluation of the universal CD40-targeting aptamer RCA adjuvant platform as a bivalent system 2. Comprehensive identification of the genetic factors in Salmonella Typhimurium required for horizontal transmission What was accomplished under these goals? We established a chicken gut colonization model using 4 different serotypes of Salmonella enterica (S. Typhimurium, S. Enteritidis, S. Heidelberg, and S. Kentucky). These challenge models will be used for evaluation of the efficacy of the vaccination with the RCA complex we developed. We developed a rolling circle amplification (RCA) product that has a specific binding affinity to various Salmonella enterica serotypes based on published Salmonella-specific aptamers. This RCA product, called 3Sal-RCA, was used as one component in the vaccine complex. We developed 3 different versions of DNA-based adjuvant based on RCA that activates macrophage through CD40 targeting. We established a macrophage activation assay using the HD-11 cell line. This assay has been used to evaluate the capacity of CD40 activation by the RCA vaccine complexes. We found RCA vaccine complex version 2 and 3 activates HD-11 cells significantly as compared to negative controls. We also started Tn-seq experiment to identify Salmonella Typhimurium genes required for survival in chicken feces.
Publications
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