1a. Objectives (from AD-416):
The overall objective of this project is to advance the development of technologies for detecting toxins that impact food safety and food defense and to determine their stability and bioavailability. Specifically, the project will focus on the following four objectives: Objective 1: Advance the development of structure- and activity-based detection methods for protein toxins. Subobjective 1A: Develop new antibodies (Abs) to botulinum neurotoxin (BoNT) serotype F, with serotypes C, D, and G as secondary priorities. Subobjective 1B: Determine the impact of different types of accessory proteins on the detection of BoNTs. Subobjective 1C: Develop activity-based detection methods for staphylococcal enterotoxin (SE) serotype E. Subobjective 1D: Develop monoclonal antibodies (mAbs) to Shiga toxin (Stx) subtypes and variants, including those from non-E. coli. Objective 2: Advance the development of detection methods for non-bacterial toxins. Subobjective 2A: Develop new detection methods for plant-derived protein toxins such as abrin. Subobjective 2B: Develop new detection methods for mushroom toxins such as amatoxins. Objective 3: Assess foodborne risks through examination of toxin stability and bioavailability in relation to intrinsic and extrinsic stresses. Subobjective 3A: Use activity-based assays to assess impact of food processing, matrices and accessory proteins on toxin activity. Subobjective 3B: Determine the factors that affect the bioavailability of toxins using rodent bioassay. Objective 4: Advance the development of instrumental, portable, and field-deployable testing methods. Subobjective 4A: Develop platforms such as optical array technologies to detect toxins. Subobjective 4B: Utilize instrumental methods to detect toxins based on mass spectra and/or other physicochemical characteristics.
1b. Approach (from AD-416):
Objective 1 has 2 general approaches: (1) Exploit ELISA and related technologies because of their versatility, robustness, and sensitivity. The mAbs developed for immunoassay will also be useful for sample preparation and for establishing design criteria for protective antibodies with clinical utility. (2) Develop activity assays for Stx2 variants and SEs. Activity assays will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are expected in processed food samples. Both approaches address practical analytical problems. The following hypotheses will be tested:(a) High affinity mAbs and recombinant Abs for BoNT serotypes and NAPs as analytical targets will provide useful reagents for ELISA and sensor methodology. (b) Neurotoxin-associated proteins (NAPs) influence the physicochemical properties of BoNT complexes, and their ease of detection in food matrices. These effects depend on the types of accessory proteins present. (c) A cell line can be engineered to provide a cell-based activity assay for SEE to measure active toxin in food matrices to replace bioassay and improve upon structure-based immunoassay. (d) New mAbs will be able to distinguish new subtypes of Stx1 or Stx2 produced by non-E. coli bacteria such as Enterobacter cloacae. Objective 2 will exploit immunoassays, especially ELISA and related technologies (as in Objective 1) and also develop and utilize activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation. The following hypotheses will be tested: (a) High affinity variant-specific mAbs will provide useful reagents for ELISA and sensor methodology for detecting nonbacterial protein toxins like abrin. (b) High affinity toxin-specific and group-specific mAbs will provide useful reagents for ELISA and sensor methodology for mushroom toxins. Objective 3 will exploit activity assays and rodent bioassay to better define the vulnerabilities of our food supply and the analytical needs. The following hypotheses will be tested: (a) Food processing conditions, food matrices, and accessory proteins impact toxin activity. (b) The oral bioavailabilities of BoNT and abrin vary among subtypes of toxin and state of the toxin (pure, complexed, crude) and depend on the food matrix. Objective 4 will exploit instrumental and portable technologies for toxin detection. Some of these technologies will will utilize binding molecules and activity assays developed under Objectives 1 and 2. To advance the development of instrumental and field-deployable testing, the following hypotheses will be tested: (a) A robust cell-based activity assay for SEE in a food matrix can be developed using a small fluorescence-detecting charge-coupled device to read data. (b) Mass spectral data and other physicochemical properties are useful for detection of toxin proteins and peptides. Contingency plans are built into the Approach for each objective and sub-objective. For example, contingencies for Objective 1 include use of alternative immunogens, sample preparation strategies, and assay formats.
3. Progress Report:
Under Objective 1A, following immunization and cell fusion experiments with recombinant derived peptide fragments of botulinum neurotoxin serotype F (BoNT/F), Researchers in Albany, California isolated nine monoclonal antibodies (mAb). The binding domain of each mAb is localized on the carboxyl terminal domain of BoNT/F (N1087—N1278). Peptide antigens for nontoxic associated proteins of BoNT/A, /B, and /E were also produced and used to generate monoclonal antibodies (mAbs). The recombinant peptides for BoNT/E associated peptides were produced in collaboration with researchers at the University of the Pacific and used by ARS researchers to generate tentative mAbs to P47, OrfX-1 and OrfX-2 associated BoNT/E proteins. Under Objective 1C, progress was made to develop activity-based detection methods for staphylococcal enterotoxin serotype E (SEE). SEE is the leading causes of foodborne illness outbreaks. Researchers in Albany, California developed and evaluated a cell proliferation assay for SEE activity based on mouse splenocytes. The detection limit of this assay was 10 ng/mL of SEE, which is about one order of magnitude less sensitive than a typical enzyme-linked immunosorbent assay (ELISA), which cannot discern active from inactive toxin, and two orders of magnitude more sensitive than in vivo kitten or monkey assays. Under Objective 1D, progress was made to develop antibodies to Shiga toxin subtypes from E. coli and non-E. coli sources. Shiga toxin-producing E. coli (STEC) are known to produce two types of Shiga toxins (Stxs), Stx 1 and Stx 2. Each type is further divided into subtypes, which have the potential to cause disease. Five new mAbs against Stx1d were newly developed. Four mAbs against Stx1e, a new subtype of Stx1 produced by an Enterobacter cloacae strain isolated from a human clinical sample were produced last year, but all of them bind to the A-subunit of the toxin. ARS researchers developed two new mAbs against the B-subunit of Stx1e. The new mAbs against the Stx1d and Stx1e were incorporated into a commercial assay. The new Stx1 ELISA not only increased the detection sensitivity to Stx1d, but also detected Stx1e with greater sensitivity. Both toxins were not previously detectable by any existing commercial Stx assays. Under Objective 2A, progress was made to develop mAbs against abrin produced by rosary pea plant. Abrin has the potential to pose a severe threat to both human and animal health and is listed as a select agent by United States Department of Health and Human Services. However, very limited reagents are available for detection. Seven new mAbs were developed and a sandwich ELISA capable of detecting abrin holotoxin was established using mAbs against the A- and B-subunits, respectively. The limit of detection of the assay was below 1 nanogram per milliliter (ng/mL) in Phosphate-Buffered Saline (PBS), non-fat milk and whole milk, which is well below the concentrations that would pose a health concern. A collection of abrin mAbs was also obtained via an interagency reimbursable agreement with the Department of Homeland Security. The mAbs were propagated, expanded, stored in a cryogenic database, affinity purified, and antibody pairs identified that are useful for capture ELISA. Detection limit for Abrin is in the low ng/mL range. Additional characterization of these mAbs is underway. Under Objective 2B, researchers developed new reagents for the detection of mushroom toxins, or amatoxins. Since amanitin toxins are too small to elicit an immune response, conjugation of toxin to a carrier protein was necessary to generate useful immunogens. Conjugation of ß-amanitin using two distinct chemistries, a mixed anhydride method and linkage via cyanuric chloride, has been completed. Additionally, three carrier proteins were used resulting in six hapten conjugates. The presence of ß-amanitin was confirmed using spectral scans and commercial antisera. Immunizations and initial antibody screens are in progress. Chemical toxoids also have been synthesized and are being used as immunogen. Under Objective 3A, progress was made to use activity-based assays to assess impact of food processing, matrices and accessory proteins on toxin activity. Applying an in vitro cell based assay for toxin activity that was previously developed by ARS researchers in Albany, California, the stability of SEE to heat treatment was evaluated in reconstituted milk and in PBS, demonstrating that milk has a protective effect on the heat inactivation of SEE. Under Objective 3B, the bioavailability of abrin after pH (2-9) or heat treatment (99, 85, 80, 74 and 63°C) was evaluated using both the in vitro cell free translation (CFT) assay and mouse bioassays. Abrin was resistant to pH treatments and remained active as demonstrated in both in vitro and mouse bioassays. Abrin was sensitive to heat treatment in temperatures above 63°C when tested in the mouse bioassays. However, heat treatment of abrin showed temperature dependent sensitivity in the in vitro CFT assay. These seemingly contradictory results illustrate the need for in vivo mouse bioassays to validate in vitro results. Also under Objective 3B, in determining factors that can affect the bioavailability of botulinum neurotoxins (BoNT), researchers evaluated the effect of probiotic bacteria on the intestinal binding and absorption of BoNTs. Probiotic microorganisms have been extensively studied for their beneficial effects in protection from allergens, pathogens, and toxins. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. Researchers also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies. In collaboration with researchers at the Kech Graduate Institute in Claremont, California, ARS researchers tested the effects of the drug Bithionol on BoNT oral uptake and showed that it inhibited mouse oral intoxication, even after intake of lethal doses of toxin. Bithionol is a Food and Drug Administration approved drug that inhibits caspases, host proteins that are used in the disease process of many toxins and pathogens. Bithionol reduced the intoxicating effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, ricin, and Zika virus. This study showed that identification and targeted inhibition of host proteins that mediate multiple disease pathways could lead to the discovery of new broad-spectrum therapies. Under Objective 4A, progress was made to develop platforms such as optical array technologies to detect toxins. A low cost charge-coupled device (CCD) camera was developed and evaluated for the assay of active SEE. Using this device, researchers were able to measure SEE concentrations over an 8 log range and with a sensitivity down to 100 femtograms (fg)/mL which is a billion times more sensitive than the comparable in vivo assays for SEE toxin activity using monkeys or kittens. The assay was verified in white grape and peach mango juices as well as in apple cider. The low cost camera device should improve the availability of toxin testing where resources are limited. Objective 4b was completed last year. A liquid chromatography/mass spectrometry (LC/MS) method to detect shiga toxins was developed. This method detected as low as 10 attomoles of toxin in a human serum matrix illustrating its potential use in clinical settings. In collaboration with the Department of Homeland Security, hybridoma cell lines producing monoclonal antibodies to the following toxins: aflatoxin; ochratoxin; diphtheria toxin; cholera toxin; abrin; and ricin were obtained, expanded, cryogenically stored in an in-house database, and antibody was produced and affinity purified for each line. A collection of purified monoclonal antibodies were obtained to the following agents: Burkholderia mallei, B. pseudomallei, Ebola virus, Francisella tularensis, Marburg virus, Staphylococcus aureus enterotoxins A and B; Microcystine-LR; ochratoxin A; ochratoxin B; T-2 toxin; tetanus toxin; and thermo labile toxin E. coli.
1. Development of new sensitive detection assay kits for all known subtypes of shiga toxins. Shiga toxins (Stx) are the main disease factors for foodborne illnesses caused by pathogenic Escherichia coli. Many new Stx subtypes, including an atypical one made by Enterobacter cloacae have recently been identified. Currently available detection assays however, do not recognize all subtypes of shiga toxins, especially those of Stx1d and 1e. ARS researchers in Albany, California developed new monoclonal antibodies against Stx1d and 1e. The technology was transferred to a commercial partner and a new sensitive detection kit capable of detecting all subtypes of Stx1 is now available. This new Stx1 detection kit should be useful for reducing product recalls and disease mistreatment due to failure of detecting less common but clinically important subtypes of Stxs.
2. In vitro method to detect and quantify active staphylococcal enterotoxin type D (SED) as an alternative to animal bioassays. Food poisoning caused by staphylococcal enterotoxins is among the leading causes of food-borne outbreaks. The current method for detection of enterotoxins activity is an emetic in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals and is impractical to test on a large number of samples. ARS researchers in Albany, California developed a robust and rapid cell-based assay using a genetically engineered T cell-line expressing the luciferase reporter gene in combination with a B-cell line for presentation of the toxin to the engineered T cell-line. Active staphylococcal enterotoxin type D produces a bioluminescence response that is dependent on toxin concentration. As little as 100 ng/mL of active SED can be detected, providing a 100,000 fold improvement in sensitivity compared to the monkey and kitten bioassays. This new method is an economical, rapid, and effective alternative to current detection standards.
3. Identification of probiotic bacteria that inhibit botulinum neurotoxin (BoNT) intestinal cell binding. Probiotic microorganisms have been extensively studied for their beneficial effects in protection from allergens, pathogens, and toxins. ARS researchers in Albany, California evaluated the effect of probiotic bacteria on the intestinal binding and absorption of BoNT serotype A. BoNTs are some of the most poisonous natural toxins known to man and are threats to public health and safety. Several probiotics that were tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A intestinal uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. These results show, for the first time, that probiotic treatment can inhibit BoNT/A binding and internalization in intestinal cells and may lead to the development of new therapies.
4. New antibodies developed against a rare subtype of shiga toxin 2. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogenic bacteria that produces many subtypes of shiga toxins (Stx) that are not well characterized and are difficult to detect with currently available detection assays. ARS researchers in Albany, California developed new toxoids for shiga toxin 2 (Stx2f) that were used as immunogens for producing high-affinity monoclonal antibodies. Stx2f was characterized and shown to be more acid and heat stable compared to common Stx2 types. Stx2f also exhibited strong binding to a model intestinal receptor suggesting that Stx2f is a pathogenicity factor with great relevance to foodborne infection. New monoclonal antibodies have been developed for the sensitive detection of Stx2f that are now patented (U.S. Patent No. 9,513,287 B1) and licensed for use in a commercially available detection assay kit. This technology expands current detection and diagnosis capabilities for Stx leading to better prevention and therapeutic outcomes.
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