The Biothreat toxins include the most lethal agents ever described. Nanogram amounts of aerosolized or injected toxins cause death. To prevent loss of life, detection of biothreat toxin must be made rapidly, accurately and with exquisite sensitivity. In addition, an ideal diagnostic should be stable and simple enough for individuals with limited medical training, such as first responders (police and fire-fighters) to use on site. Current """"""""gold standard"""""""" detection assays are time consuming and sample dependent, and fall far short of the ideal. Our preliminary studies have shown that glycoconjugate ligands mimicking the natural toxin receptor can be used for rapid and precise detection of closely related toxins. As proof of principle, we have developed tailored carbohydrates that can discriminate between the two antigenic forms of Shiga toxin, Stx1 and Stx2. We propose to extend these studies to develop diagnostic and therapeutic products.
In Aim 1, we will develop 2nd and 3rd generation toxin ligands specific for Shiga, Ricin, Botulinum, Clostridium perfingens epsilon toxins and Staphylococcus enterotoxin B (SEB). These ligands will maximize cooperative binding and optimize differentiation between variants in each toxin class.
In Aim 2 we will develop and evaluate different diagnostic platforms. Biotin will be covalently linked to the ligands. Conjugation to prefabricated streptavidin coated microtiter plates yields a glycoconjugate microarray cartridge, ideal for rapid testing of toxins in a point-of-care setting.
In Aim 3, we will validate and perform pre-clinical evaluation of lead compounds from Aim 1. We will quantify the effectiveness of the microarray diagnostics and their potential as therapeutics in tissue culture, in mouse models, and in human specimens ex vivo. Sensitivity, specificity, rapidity, ruggedness, ease-of-use and cost-effectiveness will be evaluated to establish proof of principle.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI075498-05
Application #
8100185
Study Section
Special Emphasis Panel (ZAI1-LW-M (M1))
Program Officer
Hall, Robert H
Project Start
2007-08-01
Project End
2014-04-30
Budget Start
2011-08-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2011
Total Cost
$400,068
Indirect Cost
Name
University of Cincinnati
Department
Genetics
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Karve, Sayali S; Pradhan, Suman; Ward, Doyle V et al. (2017) Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli. PLoS One 12:e0178966
Pellino, Christine A; Karve, Sayali S; Pradhan, Suman et al. (2016) AB5 Preassembly Is Not Required for Shiga Toxin Activity. J Bacteriol 198:1621-30
Karve, Sayali S; Weiss, Alison A (2014) Glycolipid binding preferences of Shiga toxin variants. PLoS One 9:e101173
Yosief, Hailemichael O; Weiss, Alison A; Iyer, Suri S (2013) Capture of uropathogenic E. coli by using synthetic glycan ligands specific for the pap-pilus. Chembiochem 14:251-9
Yosief, Hailemichael O; Iyer, Suri S; Weiss, Alison A (2013) Binding of Pk-trisaccharide analogs of globotriaosylceramide to Shiga toxin variants. Infect Immun 81:2753-60
Guo, Xuefei; Kulkarni, Ashish; Doepke, Amos et al. (2012) Carbohydrate-based label-free detection of Escherichia coli ORN 178 using electrochemical impedance spectroscopy. Anal Chem 84:241-6
Gallegos, Karen M; Conrady, Deborah G; Karve, Sayali S et al. (2012) Shiga toxin binding to glycolipids and glycans. PLoS One 7:e30368
Fuller, Cynthia A; Pellino, Christine A; Flagler, Michael J et al. (2011) Shiga toxin subtypes display dramatic differences in potency. Infect Immun 79:1329-37
Conrady, Deborah G; Flagler, Michael J; Friedmann, David R et al. (2010) Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2. PLoS One 5:e15153
Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S (2010) Detection of carbohydrate binding proteins using magnetic relaxation switches. Anal Chem 82:7430-5

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