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

2022 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 Toxins and Allergens that Impact Food Safety and Food Defense,” which started on December 28, 2020, and continues research from the project 2030-42000-049-000D, “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 in Albany, California, generated inactive botulinum neurotoxin (BoNT) serotypes BoNT/C and BoNT/DC. These inactive toxins were used to immunize mice and hybridoma cell lines secreting monoclonal antibodies were produced. Six monoclonal antibodies recognized BoNT/C and five monoclonal antibodies detected BoNT/DC. These antibodies are critical new reagents to complement existing detection assays and develop new diagnostic detection assays currently not commercially available. In support of Sub-objective 1A, ARS researchers produced a chimeric BoNT serotype H (F/A) toxoid. Mice were immunized with this inactivated toxoid and screened for hybridoma cell lines producing monoclonal antibodies. Sixteen monoclonal antibodies were generated that detected BoNT/H (FA) as well as three other BoNTs important for causing human botulism. These antibodies are critical new reagents due to their potential ability to detect multiple BoNT serotypes at the same time using a single antibody in novel diagnostic detection assays currently not commercially available. In support of Sub-objective 1B, ARS researchers implemented the contingency plan to produce a Vvha toxoid, a Vibrio vulnificus cytolysin. Mice were immunized with this inactivated toxoid and six monoclonal antibodies were generated after antibody screening and hybridoma fusion. These six monoclonal antibodies were able to detect Vvha. These antibodies are critical new reagents for the development of new diagnostic detection assays currently not available. In support of Sub-objective 1D, ARS researchers developed an ex vivo assay for the detection of Streptococcal Pyrogenic Exotoxins (SPEs). Murine splenocytes treated with SPE-B caused a significant increase in the number of cells and a measurable cytokine release in response to biologically active toxin. Researchers will continue to optimize the ex vivo assay and compare it to other cell-based assays in development, thus reducing the need for in vivo assays currently in use. Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus (S. aureus) is one of the most potent bacterial superantigens with profound toxic effects on the immune system and can cause food poisoning. The current diagnostic methods for SEB are mainly based on traditional monoclonal antibodies that have limited clinical use and the hybridoma cell lines producing these monoclonal antibodies can lose their ability to make and secrete these critical antibodies over time. In support of Sub-objective 1E, ARS researchers developed novel nanobodies capable of being used for detection and neutralization of Staphylococcal enterotoxin B (SEB) in foods. The development of these novel nanobodies are critical resources for developing improved diagnostic tools to detect this potent toxin. In support of Sub-objective 1F, ARS researchers generated and prepared recombinant Pru du 8 as an antigen. This purified protein will be used to immunize mice and generate monoclonal antibodies. In support of Objective 3, ARS researchers determined the antibody protection ability of monoclonal antibodies to BoNT/G using the in vivo mouse bioassay. Ten monoclonal antibody combinations were able to protect mice from death when injected with a mean lethal dose (MLD50) of 10,000 MLD50, a dose that is 10,000 times more than needed to cause death in a single mouse, thus showing the significant protective effect of these antibodies. These antibodies are significant critical reagents for neutralizing BoNT/G since there are no commercially available therapies for BoNT/G intoxication. In support of Objective 3, ARS researchers investigated the effect of a broad-spectrum and reversible antibiotic guanylhydrozone disinfectant developed in-house on Escherichia coli (E. coli) to determine if any antimicrobial resistance is generated due to the pervasive use of antibiotics leading to ecotoxicity and antibiotic resistance. The results showed that no resistance developed to the disinfectant after treatment for 30 days on 37 Shiga toxin-producing E. coli (STEC) strains, nor induced Shiga toxin production. Importantly, disinfectant use significantly reduced expression of genes responsible for ribosome and flagellar assembly suggesting that the mode of action may be through inhibiting protein synthesis and bacterial mobility. This study will allow a better understanding of the mode of the disinfectant action and make the disinfectant application more efficient.

Accomplishments

Review Publications
Rasooly, R., Howard, A., Balaban, N., Hernlem, B.J., Apostolidis, E. 2022. The effect of tannin-rich witch hazel on growth of probiotic Lactobacillus plantarum. Antibiotics. 11(3). Article 395. https://doi.org/10.3390/antibiotics11030395.
Rasooly, R., Do, P.M., Hernlem, B.J. 2022. Ex vivo and in vitro methods for detection of bioactive staphylococcal enterotoxins. In: Ossandon, M.R., Baker, H., Rasooly, A., editors. Biomedical Engineering Technologies. Volume 1. New York, NY: Humana Press. p. 237-255. https://doi.org/10.1007/978-1-0716-1803-5_13.
Failla, M., Lee, J., Rasooly, R., Apostolidis, E. 2022. Evaluation of a witch hazel extract for the potential prebiotic and protective effect on select Lactiplantibacillus plantarum (prev. Lactobacillus plantarum) strains. Frontiers in Nutrition. 9. Article 874666. https://doi.org/10.3389/fnut.2022.874666.