Research Project: Technologies for the Detection of Bacterial and Plant Toxins and Allergens that Impact Food Safety and Food Defense

Location: Foodborne Toxin Detection and Prevention Research

2024 Annual Report

Objectives
Project scientists have extensive previous experience and success in the development of antibody reagents for the sensitive detection of Shiga toxins, botulinum neurotoxins, abrin, Zika, amatoxins, and colistin resistance. In addition, they have successfully developed immunoassays, novel cell-based and biosensor detection methods, as well as identified antibody combinations, receptor mimics, and drugs that reduce toxin toxicity. The proposed plan below will build on our past accomplishments and expand our research agenda into new toxins, bacterial virulence factors, and food allergen antigen targets. Objective 1: Develop reagents to detect bacterial and non-bacterial toxins. • Sub-objective: 1.A: Develop high affinity mAb that recognize multiple serotypes of BoNT. • Sub-objective: 1.B: Develop new monoclonal antibodies (mAbs) against the analytical targets of V. vulnificus: the toxin RtxA1/MARTXVv with secondary priorities against the cytolysin/hemolysin Vvha effectors of the two Type 6 Secretion Systems. • Sub-objective: 1.C: Develop technology for detection of biologically active Staphylococcal enterotoxin type B (SEB) in food. • Sub-objective: 1.D: Develop methodology for detection of biologically active Streptococcal Pyrogenic Exotoxin (SPEs) using cell-based assays. • Sub-objective: 1.E: Develop nanobodies for Stxs and SEB. • Sub-objective: 1.F: Develop novel antibodies to almond allergens. Objective 2: Advance the development of instrumental, portable, and field-deployable testing methods for bacterial and non-bacterial toxins. • Sub-objective: 2.A: Develop new immunoassays such as ELISAs, lateral flow devices, and cell-based biosensor assays that recognizes multiple BoNT serotypes. • Sub-objective: 2.B: Develop new immunoassays such as ELISAs and lateral flow devices using mAbs to RtxA1/MARTXVv or cytolysin/hemolysin Vvha. • Sub-objective: 2.C: Develop new multi-spectra analysis technology (optical array) to detect multiple pathogens and/or toxins simultaneously. • Sub-objective: 2.D: Using antibodies developed in Objective 1E and 1F, develop immunoassays such as ELISA, lateral flow immunoassays, or other portable assays for Stx, SEB, and almond allergens. Objective 3: Assess and utilize monoclonal antibodies, small molecule drugs, and natural compounds for use in toxin neutralization. • Sub-objective: 3.A: High affinity multi-serotype mAbs can neutralize intoxication by human BoNT serotypes (A,B,E,F). • Sub-objective: 3.B: High affinity mAbs to Vibrio vulnificus virulence factors for toxin neutralization. • Sub-objective: 3.C: Neutralize Stxs and SEB using Nbs.

Approach
1A. Botulinum neurotoxin (BoNT) detection is complicated by its many variants and the lack of analytical tools to detect them. The generation of high-affinity multi-serotype monoclonal antibodies (mAbs) will fulfill this need. 1B. Vibrio vulnificus is the causative agent for over 95% of seafood-related deaths in the U.S. The development of high-affinity mAbs against RTXA1/MARTXVv is vital. 1C. Staphylococcal enterotoxin type B (SEB) is highly toxic, thermal stable, and involved in foodborne outbreaks. Generation of SEB-specific cell-based assays based on its superantigenic activity will allow for the detection of active toxin in food. 1D. Streptococcal Pyrogenic Exotoxins (SPEs) are toxins that are involved in foodborne outbreaks. The development of SPE-specific reporter cell lines will allow for active toxin detection in food. 1E. Conventional antibodies are large and hence limits its application. Development of nanobodies to Stxs and SEB will eliminate these obstacles and expand capability. 1F. Almond nut allergies are of grave concern to the almond industry and consumers. The development of high affinity monoclonal antibodies to new almond food allergens will provide vital reagents that are lacking. 2A. Few assays exist that can detect multiple BoNT serotypes simultaneously in a rapid, sensitive, and user-friendly format. Using the multi-serotype mAbs developed in sub-objective 1A, an updated, fast, sensitive, and easy to use detection method (ELISA) or other immunoassays will be developed to detect multiple BoNTs in food. 2B. The lack of commercially available ELISAs, lateral flow devices (LFDs), or biosensor assays for the rapid and portable detection of V. vulnificus or its toxins is of great concern. High-affinity mAbs against its main toxin will be used to develop ELISAs and/or lateral flow devices to detect toxin contamination in food. 2C. The current patented multi-spectra array optical detector has multiplexing limitations. The development of a new software method to separate out discrete spectral differences amongst different fluorophores will enhance its multiplexing capability. 2D. Antibodies developed in sub-objective 1E and 1F will be used in diagnostic assays such as ELISAs, lateral flow assays, and other portable immunoassays to detect Stx, SEB, and almond food allergens. 3A. Immunotherapies to human botulism are few, costly, limited, and have potential adverse effects. The multi-serotype mAbs will be tested for toxin neutralization activity against human botulism serotypes using in vivo mouse bioassays and in vitro cell or activity-based assays. 3B. There is a lack of available immunotherapies to treat V. vulnificus infections. The mAbs from sub-objective 2B will be tested for toxin neutralization activity using in vivo mouse bioassays and/or in vitro cell culture models. 3C. Neutralizing nanobodies (Nbs) have great therapeutic potential in comparison with conventional immunoglobulin therapy. Nbs will be screened using cell-based assays (Stxs, Vero cell based screen for toxicity; SEB, specific SEB-mammalian reporter cell line). Contingency plans are built into the Approach for each objective and sub-objective.

Progress Report
This report documents progress for project 2030-42000-053-000D, “ Technologies for the Detection of Bacterial and Plant and Allergens that Impact Food Safety and Food Defense”, which started in December 2020. In support of Sub-objective 1A, ARS researchers in Albany, California, evaluated a panel of seven monoclonal antibodies (mAbs) targeted against botulinum neurotoxin serotype F (BoNT/F), one of the major human disease causing serotypes. Researchers also generated ascites fluids and purified potential multi-serotype mAbs targeted against BoNT/H (BoNT/FA). Preliminary optimization studies were performed and further optimization is required. These new antibodies are critical reagents that will compliment existing and/or future development of new diagnostic assays currently not commercially available. For Sub-objective 1B, ARS researchers in Albany, California, characterized a panel of twelve monoclonal antibodies (mAbs) targeted against Vvha, a major toxin that causes mammalian cell death. Vvha, is one of the contributing virulence factors of Vibrio vulnificus, and this type of bacteria causes disease after accidental ingestion of oysters/seafood contaminated with V. vulnificus or exposure of wounds to V. vulnificus contaminated water sources. These mAbs were specific to Vvha and are critical new reagents to incorporate in diagnostic detection assays that are not currently available. In support of Sub-objective 1C, ARS researchers in Albany, California, evaluated ex vivo and in vitro assays for Staphyloccal enterotoxin B (SEB) detection in foods. SEB is one of the most potent bacterial superantigens with profound toxic effects on the immune system and can cause food poisoning. The assays were able to detect SEB in foods but were not able to distinguish between biologically active and inactive SEB. Further optimization may improve the performance of the assay. For Sub-objective 2A, ARS researchers in Albany, California, developed a direct enzyme-linked immunosorbent assay (ELISA) to detect multiple serotypes of botulinum neurotoxins (BoNTs). Eight multi-serotype monoclonal antibodies were evaluated for their performance and all eight were able to bind to BoNT/A, BoNT/F, BoNT/E, and BoNT/H (BoNT/FA) in a direct ELISA. Preliminary experiments using purified antibodies to determine capture-detector antibody pairs using these mAbs were partially successful but with high limits of detection. Direct ELISA optimizations using the new BoNT/F mAbs were promising. Future optimization with these mAbs in conjunction with commercially available BoNT antibodies will be explored. Once optimization and determination of the best capture-detector antibody pairs may yield a sensitive ELISA assay to detect all know BoNT serotypes will critically fill in the gap in any commercially available multi-serotype BoNT ELISAs. In support of Sub-objective 2B, ARS researchers in Albany, California, developed a preliminary capture-capture ELISA against Vibrio vulnificus Vvha toxin. The assay utilizes USDA in-house Vvha monoclonal antibodies as capture antibodies and a commercially available polyclonal Vvha-HRP as a detector. Further optimization will improve the performance of this ELISA and increase sensitivity. The development of a new detection assay against a critical toxin from Vibrio vulnificus will improve food safety and public health. For Sub-objective 2C, ARS researchers in Albany, California, optimized the technology and developed software to detect multiple targets at the same time in the same sample. The researchers built on previously developed Python code for the analysis of multi-color assay images and applied the software to analyze images of multi-well plates mimicking a high-throughput workflow needed for surveillance. Additionally, an improved plate holder for fluorescence imaging was developed in conjunction with a sensitive Complementary Metal Oxide Semiconductor (CMOS) camera. Further optimization may yield improved performance and the capability to detect simultaneous targets of interest in a high-throughput format important for food safety surveillance programs. In support of Sub-objective 2D, ARS researchers in Albany, California, continued developing methods for detection of Shiga toxin 2 (Stx2) with collaborators at Smiths Detection. A rapid, sensitive, and potentially portable assay capable of identifying Shiga toxin-producing E. coli (STEC) by detecting Stx2 was developed using a B-cell based biosensor. The biosensor was able to detect all Stx2 subtypes associated with human diseases and had a Limit of Detection (LOD) of 0.1 ng/mL for Stx2 in buffer. When applied to STEC-spiked milk, lettuce, and beef, it was able to identify samples inoculated with 0.4 cfu/g or 0.4 cfu/mL of STEC. The rapid assay time (3 minutes) and small volumes needed (200 microliters) makes this detection technology a potent qualitative tool in environmental and food safety surveillance programs.

Accomplishments
1. Development of a rapid and sensitive CANARY biosensor for the detection of Shiga toxin-producing E. coli. Shiga-toxin producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Current testing methods for STEC generally include enrichment, cell plating, and genomic sequencing which takes time to complete thus delaying diagnosis and treatment. ARS researchers in Albany, California, developed a rapid (within 3 minutes), sensitive (100 pg/mL), and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the Cellular Analysis and Notification of Antigen Risks and Yields (CANARY) B-cell based biosensor technology. This technology could be highly useful for environmental and food safety surveillance programs.

2. Detection of biologically active Streptococcal pyrogenic exotoxin B in milk. Streptococcal pyrogenic toxins (SPEs) are some of the most commonly found food contaminants and people get ill eating these contaminated foods. Assays to detect and quantify Streptococcal pyrogenic exotoxin serotype B (SPE-B) ideally respond only the active form of the toxin and this usually employs animal testing. ARS researchers in Albany, California, developed a cell-based assay for active SPE-B quantification based on murine splenocyte proliferation and Il-2 cytokine secretion. This assay detected biologically active SpeB in milk after pasteurization for 30 minutes at 63°C. This assay allows for the rapid and sensitive detection of contaminated foods thus improving food safety.

3. Detection of toxic mushrooms using antibody enriched Liquid-chromatography-high-resolution mass spectrometry (LC-HRMS). Amatoxins are toxic substances found in several poisonous mushroom species including the well-known “Death Cap” mushroom. Ingestion of these toxic substances can lead to liver failure and death. Current clinical detection methods usually require extraction and detection of amatoxin from urine of suspected amatoxin ingestion patients. ARS researchers in Albany, California, along with collaborators at Saarland University, Germany, developed an affinity-enriched liquid-chromatography-high resolution mass spectrometry (LC-HMS) that was able to detect amatoxin in urine with a limit of detection of 1 ng/mL. The assay is valuable for the rapid and sensitive detection of amatoxins needed in emergencies since amatoxin ingestion results in symptoms approximately 12 hours after ingestion and amatoxins may already be eliminated in plasma and concentrated in urine. Rapid detection allows for earlier therapeutic intervention and improved outcome.

Review Publications
Kellnerova, S., Huber, S., Massri, M., Fleischer, V., Losso, K., Sarg, B., Kremser, L., Talasz, H., He, X., Varrone, E., Brigotti, M., Ardissino, G., Orth-Holler, D., Wurzner, R. 2023. Enzymatic cleavage of Stx2a in the gut and identification of pancreatic elastase and trypsin as possible main cleavers. Microorganisms. 11(10). Article 2487. https://doi.org/10.3390/microorganisms11102487.
Tam, C.C., Wang, Y., Du, W.N., Flannery, A.R., He, X. 2024. Development of a rapid and sensitive CANARY biosensor assay for the detection of Shiga toxin 2 from Escherichia coli. Toxins. 16(3). Article 148. https://doi.org/10.3390/toxins16030148.
Carter, M.Q., Quinones, B., He, X., Pham, A.C., Carychao, D.K., Cooley, M., Lo, C., Chain, P.S., Lindsey, R.L., Bono, J.L. 2023. Genomic and phenotypic characterization of Shiga toxin-producing Escherichia albertii strains isolated from wild birds in a major agricultural region in California. Microorganisms. 11(11). Article 2803. https://doi.org/10.3390/microorganisms11112803.
Carter, M.Q., Quinones, B., Laniohan, N.S., Carychao, D.K., Pham, A.C., He, X., Cooley, M. 2023. Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California. Frontiers in Microbiology. 14. Article 1214081. https://doi.org/10.3389/fmicb.2023.1214081.
Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2023. A sensitive, cell-based assay for measuring low-level biological activity of a-amanitin. International Journal of Molecular Sciences. 24(22). Article 16402. https://doi.org/10.3390/ijms242216402.
Zheng, Q., Chen, Z., Yan, W., Wang, H., Tian, W., Feng, D., Yue, L., Qi, W., He, X., Kong, Q. 2023. TMT-based quantitative proteomic and scanning electron microscopy reveals biological and morphological changes of Staphylococcus aureus irradiated by electron beam. LWT - Food Science and Technology. 184. Article 114977. https://doi.org/10.1016/j.lwt.2023.114977.
Gao, B., Zhu, H., Liu, Z., He, X., Sun, J., Li, Y., Wu, X., Pehrsson, P.R., Zhang, Y., Yu, L. 2024. Chemical compositions of Scutellaria baicalensis Georgi. (Huangqin) extracts and their effects on ACE2 binding of SARS-CoV-2 spike protein, ACE2 activity, and free radicals. International Journal of Molecular Sciences. 25(4). Article 2045. https://doi.org/10.3390/ijms25042045.
Wu, H., Liu, Z., Zhang, Y., Gao, B., Li, Y., He, X., Sun, J., Choe, U., Chen, P., Blaustein, R., Yu, L. 2024. Chemical composition of turmeric (Curcuma longa L.) ethanol extract and its antimicrobial activities and free radical scavenging capacities. Foods. 13(10). Article 1550. https://doi.org/10.3390/foods13101550.
Varrone, E., Carnicelli, D., He, X., Grasse, M., Stampfer, K., Huber, S., Kellnerova, S., Tazzari, P., Ricci, F., Paterini, P., Ardissino, G., Morabito, S., Orth-Holler, D., Wurzner, R., Brigotti, M. 2023. Detection of cleaved Stx2a in the blood of STEC-infected patients. Toxins. 15(12). Article 690. https://doi.org/10.3390/toxins15120690.