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

2023 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.” In support of Sub-objective 1A, ARS researchers in Albany, California, tested a panel of monoclonal (mAb) antibodies targeted against a chimeric botulinum neurotoxin serotype H (BoNT/FA) for their ability to detect all BoNT serotypes. In a direct enzyme-linked immunosorbent assay (ELISA) format using non-purified hybridoma supernatants, all the antibodies detected BoNT/H as well as three other BoNT serotypes (BoNT/F, BoNT/E, and BoNT/A). Future work seeks to further optimize the multi-serotype BoNT assay for high sensitivity. Once further optimized, this new multi-serotype ELISA assay will complement current detection assays as well as replace the existing gap in detection technology due to the loss of the only commercially available BoNT multi-serotype ELISA assay. In support of Sub-objective 1F, ARS researchers in Albany, California, immunized mice with recombinant Pru du 8 as an antigen. Hybridoma cell lines were screened for expression of monoclonal antibodies that recognized Pru du 8. A panel of mAbs was generated that detected Pru du 8 in an ELISA. Further optimization will be used to incorporate these mAbs into a rapid and sensitive detection technology. In support of Sub-objective 2C, ARS researchers in Albany, California, tested and evaluated multiple microscopy deconvolution programs to develop a rapid and sensitive quantitative software for simultaneous identification of multiple targets conjugated to different fluorophores. ARS scientists wrote and developed Python computer programs that used machine learning of multiple RAW microscopic images to construct algorithms to distinguish between images labelled with identical fluorophores in three different pixel ranges (Red, Green, and Blue). Further optimization to include other fluorophores will be initiated thus allowing for the rapid and simultaneous identification and detection of multiple targets of interest in image-based detection technologies. In support of Sub-objective 2D, ARS researchers in Albany, California, characterized sixteen novel nanobodies (nbs) targeted to Staphylococcal enterotoxin B (SEB), a class B priority toxin due to its high stability, toxicity and ease of dissemination. These 10 monovalent and six divalent SEB nbs were incorporated into sensitive immunoassays that were able to detect SEB with a limit of detection (LOD) as low as 50 pg/mL in phosphate buffer saline (PBS) buffer and 190 pg/mL in milk. These novel SEB Nbs are critical novel reagents that have significantly higher stability and longer shelf-life compared to conventional antibodies making them excellent tools for sensitive, specific, and cost-effective detection and management of SEB-contaminated food products. In support of Objective 3, ARS researchers in Albany, California, developed a humanized monoclonal antibody against Shiga toxin 2 (Stx2) (Hu-mAb 2-5) and demonstrated that this humanized antibody had low immunogenicity ex vivo and high neutralizing efficacy in vivo, protecting mice from mortality and hemolytic uremic syndrome-related (HUS) tissue damage. This critical reagent may be a great tool in the treatment of people with HUS after ingestion with Stx2 contaminated food. In support of Sub-objective 3C, ARS researchers in Albany, California, characterized 16 novel nanobodies (nbs) for their neutralization activity against Staphylococcal enterotoxin B (SEB). Eleven out of the 15 SEB nbs were capable of neutralizing SEB’s super-antigenic activity. Compared to conventional antibodies, nbs are small, thermally stable, and easy to produce, making them great tools for sensitive, specific, and cost-effective detection and management of SEB contamination in food products, thus improving food safety. Natural antibiotic agents extracted from plants have been broadly used in food industries to improve food safety, quality, and shelf-life. In support of Objective 3, ARS researchers in Albany, California, in collaboration with scientists at the University of Maryland, investigated bioactive components of cinnamon and honeysuckle and their capabilities in suppressing the binding of SARS-CoV-2 spike protein to ACE2, a human protein serving as an entrance for the access of coronaviruses. This study provided new evidence that cinnamon and honeysuckle have the potential to reduce the risk of SARS-CoV-2 infection and the development of severe Covid-19 symptoms thus improving human health.

Accomplishments
1. A three-monoclonal antibody combination potently neutralizes BoNT/G in mice. Botulinum neurotoxins (BoNT) are some of the most potent toxins known to man and are both public health and food safety concerns. There are at least seven different botulinum neurotoxin serotypes (A-G) and potent neutralizing anti-toxin countermeasures to all of the serotypes do not exist. ARS researchers in Albany, California, along with collaborators at the University of California, San Francisco, developed new recombinant antibodies to BoNT/G. Ten antibody combinations protected 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. These antibodies are significant critical reagents that can neutralize BoNT/G since there are no commercially available therapeutics for BoNT/G intoxication. These antibodies will be of tremendous interest to governmental and public health agencies as well as hospitals as novel countermeasures to botulism.

2. Development and preclinical evaluation of a novel humanized Stx2 antibody. Shiga toxin producing Escherichia coli (STEC) is a major risk to food safety. Shiga toxin (Stx) is the predominant cause of the life-threatening STEC-associated hemolytic uremic syndrome (HUS). Currently, there are no Food and Drug Administration-approved therapies to treat HUS. ARS researchers in Albany, California, have developed a humanized antibody against the Stx2 and demonstrated that this humanized antibody had low immunogenicity in human whole blood cells, high Stx2 neutralizing efficacy in vitro and in vivo, protected mice from mortality and HUS-related tissue damage. This invention provides a promising therapeutic option to combat the HUS disease.

3. T-cell-receptor Vß8 for detection of biologically active streptococcal pyrogenic exotoxin type C. Streptococcal pyrogenic exotoxins (SPEs) are some of the most commonly found food contaminants and people get ill when eating these contaminated foods. Assays to detect and quantify Streptococcal pyrogenic exotoxin serotype C (SPE-C) ideally respond only to 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-C quantification in which SPE-C are presented by human B-cells to T-cells. The assay measures an increase of the IL-2 cytokine and an increase in luciferase light emission specifically to SPE-C. This is the first demonstration of a sensitive alternative assay that allows for the quantitative detection of active SPE-C. The assay allows for the rapid and sensitive detection of contaminated foods thus improving food safety.

4. Identification of nanobodies capable of detecting and detoxifying SEB. Staphylococcal enterotoxin type B (SEB) is one of the toxins responsible for staphylococcal food poisoning in humans and has been produced by some countries as a biological weapon. It is a superantigen acting by stimulating cytokine release and inflammation. There are limited therapies to treat diseases caused by SEB. ARS researchers in Albany, California, have developed novel nanobodies that can be used both for detection and neutralization of SEB. Compared to conventional antibodies, these nanobodies are small, thermally stable, and easy to produce, making them useful for cost-effective detection and management of SEB contamination in food products.

Review Publications
Fan, Y., Lou, J., Tam, C.C., Wen, W., Conrad, F., Alves, P., Cheng, L.W., Garcia-Rodriguez, C., Farr-Jones, S., Marks, J.D. 2023. A three-monoclonal antibody combination potently neutralizes BoNT/G toxin in mice. Toxins. 15(5). Article 316. https://doi.org/10.3390/toxins15050316.
Li, Y., Wu, K., Liu, Z., He, X., Sun, J., Zeng, M., Lee, S., Wang, T.T., Gao, B., Xie, Z., Wei, C., Yu, L. 2022. Effect of thermal treatments on soluble-free, soluble-conjugated, and insoluble-bound phenolic components and free radical scavenging properties of tomato seeds. ACS Food Science and Technology. 2(10):1631-1638. https://doi.org/10.1021/acsfoodscitech.2c00228.
Li, Y., Liu, Z., Zeng, M., El Kadiri, A., Huang, J., Kim, A., He, X., Sun, J., Chen, P., Wang, T.T., Zhang, Y., Gao, B., Xie, Z., Yu, L. 2022. Chemical compositions of clove (Syzygium aromaticum (L.) Merr. & L.) extracts and their potentials in suppressing SARS-CoV-2 spike protein-ACE2 binding, inhibiting ACE2, and scavenging free radicals. Journal of Agricultural and Food Chemistry. 70(45):14403-14413. https://doi.org/10.1021/acs.jafc.2c06300.
Gao, B., Zhu, L., Liu, Z., Li, Y., He, X., Wu, X., Pehrsson, P.R., Sun, J., Xie, Z., Slavin, M., Yu, L. 2023. Chemical composition of honeysuckle (Lonicerae japonicae) extracts and their potential in inhibiting the SARS-CoV-2 spike protein and ACE2 binding, suppressing ACE2, and scavenging radicals. Journal of Agricultural and Food Chemistry. 71(15):6133-6143. https://doi.org/10.1021/acs.jafc.3c00584.
Wang, Y., Hart-Cooper, W.M., Rasooly, R., Carter, M.Q., Orts, W.J., Gu, Y.Q., He, X. 2022. Effect of an eco-friendly cuminaldehyde guanylhydrazone disinfectant on Shiga toxin production and global transcription of Escherichia coli. Toxins. 14(11). Article 752. https://doi.org/10.3390/toxins14110752.
Xie, Z., Li, Y., Liu, Z., Zeng, M., Moore, J., Gao, B., Wu, X., Sun, J., Wang, T.T., Pehrsson, P.R., He, X., Yu, L. 2023. Chemical characterization of cinnamon (Cinnamomum verum J. Presl) extracts and their capacities in weakening SARS-CoV-2 spike protein binding to ACE2, inhibiting ACE2 and scavenging free radicals. Journal of Agricultural and Food Chemistry. 71:4890-4900. https://doi.org/10.1021/acs.jafc.3c00285.
Hughes, A.C., Kirkland, M., Du, W.N., Rasooly, R., Hernlem, B.J., Tam, C.C., Zhang, Y., He, X. 2023. Development of thermally stable nanobodies for detection and neutralization of Staphylococcal enterotoxin B. Toxins. 15(6). Article 400. https://doi.org/10.3390/toxins15060400.