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

2021 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 2030-42000-053-00D, “Technologies for the Detection of Bacterial and Plant Toxins and Allergens that Impact Food Safety and Food Defense,” which started on December 28, 2020, and continues research from project 2030-42000-049-00D, “Advancing the Detection of Technologies for Detecting and Determining the Stability and Bioavailability of Toxins that Impact Food Safety and Food Defense.” In support of Objective 1, ARS researchers at Albany, California, generated inactive botulinum toxins serotype BoNT/DC, and BoNT/D. These inactive toxins were used to immunize rabbits and polyclonal rabbit antibodies were generated. Two polyclonal antibodies recognized BoNT/DC and two others recognized BoNT/D specifically. These antibodies are critical new reagents for the development of new diagnostic detection assays currently not commercially available. In support of Sub-objective 1C, ARS researchers at Albany, California, used the human T-cell line, HPB-ALL, to create a new detection assay specific to Staphylococcal enterotoxin type B (SEB). The HPB-ALL cells, when paired with a human B-cell (Raji) line, responded to the presence of active SEB by secreting the cytokine interleukin-2 (IL-2). Incubation of SEB with HPF-ALL and Raji cells led to quantitative and dose-dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEB and did not detect staphylococcal enterotoxin (SE) toxin subtypes A, D, and E. This assay is 100,000 times more sensitive in comparison to the conventional ex vivo murine splenocyte method with a detection limit of 10 pg/mL.

Accomplishments
1. Investigation of mobile colistin-resistance in U.S. animal-origin food. The increasing risk of antibiotic resistance, especially of multiple mobile colistin resistance (mcr) to common last-resort antibiotics, is a public health threat. However, the prevalence of these mcr genes in U.S. meat, catfish, and poultry was previously unknown. ARS researchers at Albany, California, in collaboration with the United States Food Safety Inspection Service, screened over 5,000 domestic food samples for the presence of mcr genes using a novel enrichment method coupled to a real-time polymerase chain reaction (PCR) assay targeting mcr-1 to mcr-8. The results indicated that there was a very low prevalence of mcr-1 to mcr-8 genes present in all the meat samples tested with a final positivity rate of 0.29%. This is the first systemic and large-scale investigation of multiple mobile colistin-resistance in U.S. food animal products and the information obtained is useful for trade and food safety risk assessments.

2. Development of sensitive activity assay for Staphylococcal enterotoxin serotype B. Staphylococcal enterotoxins (SEs) are some of the most commonly found food contamination agents and people get ill when eating these contaminated foods. Assays to detect and quantify Staphylococcal enterotoxin serotype B (SEB) ideally respond only to the active form of the toxin and this usually employs animal testing. ARS researchers at Albany, California, developed a cell-based assay for active SEB quantification in which SEB is presented by human B-cells to T-cells. The assay measures a decrease in levels of the T-cell receptor Vbeta5 and an increase of the IL-2 cytokine. This is the first demonstration of a sensitive alternative assay that eliminated the use of animals for the quantitative detection of active SEB. This assay allows for the rapid and sensitive detection of contaminated foods, thus improving food safety.

Review Publications
Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2021. Human leukemia T-cell lines as alternative to animal use for detecting biologically active Staphylococcal enterotoxin type B. Toxins. 13(5). Article 300. https://doi.org/10.3390/toxins13050300.
Wang, Y., Hou, N., Rasooly, R., Gu, Y.Q., He, X. 2021. Prevalence and genetic analysis of chromosomal mcr-3/7 in Aeromonas from U.S. animal-derived samples. Frontiers in Microbiology. 12. Article 667406. https://doi.org/10.3389/fmicb.2021.667406.