Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

PubMed Central

Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M.; Zhang, Yuhua; Arnold, Beth A.; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E.; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M.

2014-01-01

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA).

PubMed

O'Hara, Joanne M; Kasten-Jolly, Jane C; Reynolds, Claire E; Mantis, Nicholas J

2014-01-01

Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design. Copyright © 2013 Elsevier B.V. All rights reserved.

Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

PubMed Central

Song, Kejing; Mize, R. Ranney; Marrero, Luis; Corti, Miriam; Kirk, Jason M.; Pincus, Seth H.

2013-01-01

Background Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood. Methodology/Principal Findings We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. Conclusions/Key Findings We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs. PMID:23638075

Immunity to Ricin: Fundamental Insights into Toxin–Antibody Interactions

PubMed Central

O'Hara, Joanne M.; Yermakova, Anastasiya

2015-01-01

Ricin toxin is an extraordinarily potent inducer of cell death and inflammation. Ricin is also a potent provocateur of the humoral immune system, eliciting a mixture of neutralizing, non-neutralizing and even toxin-enhancing antibodies. The characterization of dozens of monoclonal antibodies (mAbs) against the toxin's enzymatic (RTA) and binding (RTB) subunits has begun to reveal fundamental insights into the underlying mechanisms by which antibodies neutralize (or fail to neutralize) ricin in systemic and mucosal compartments. This information has had immediate applications in the design, development and evaluation of ricin subunit vaccines and immunotherapeutics. PMID:22113742

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies*

PubMed Central

Herrera, Cristina; Tremblay, Jacqueline M.; Shoemaker, Charles B.; Mantis, Nicholas J.

2015-01-01

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. PMID:26396190

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

PubMed Central

Leysath, Clinton E.; Ofori, Kwasi; Baldwin, Karen; Feng, Xiaochuan; Bedenice, Daniela; Webb, Robert P.; Wright, Patrick M.; Smith, Leonard A.; Tzipori, Saul; Shoemaker, Charles B.

2012-01-01

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit. PMID:22238680

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

PubMed

Mukherjee, Jean; Tremblay, Jacqueline M; Leysath, Clinton E; Ofori, Kwasi; Baldwin, Karen; Feng, Xiaochuan; Bedenice, Daniela; Webb, Robert P; Wright, Patrick M; Smith, Leonard A; Tzipori, Saul; Shoemaker, Charles B

2012-01-01

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Antibody-mediated inhibition of ricin toxin retrograde transport.

PubMed

Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

2014-04-08

Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake. In this report, however, we describe a neutralizing monoclonal antibody (MAb) against ricin's binding subunit (RTB) that not only associates with ricin after the toxin has bound to the cell's surface but actually enhances toxin uptake into host cells. Following endocytosis, the antibody-toxin complexes are then routed for degradation. The results of this study are important because they reveal a previously unappreciated role for B-subunit-specific antibodies in intracellular neutralization of ricin toxin.

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

PubMed

Yermakova, Anastasiya; Klokk, Tove Irene; O'Hara, Joanne M; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

2016-03-07

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

PubMed

Féraudet-Tarisse, Cécile; Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R; Dorner, Brigitte G; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

PubMed Central

Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R.; Dorner, Brigitte G.; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test. PMID:28700661

X Irradiation and Toxin Neutralization by Antitoxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kahn, R. L.; Kim, S. H.; Curtis, A. C.

1960-11-01

A small area of the skin of normal rabbits was exposed to 1000 r and, after a given interval, 15 units of horse serum diphtheria antitoxin were injected subcutaneously in that area and simultaneously a lethal (25 MLD) dose of homologous toxin in another area. As controls, non-irradiated rabbits of similar weight were injected with the same quantities of antitoxin and of toxin at the same time as the irradiated rabbits and under identical conditions. Five intervals following the irradiation were employed for the injection of the antitoxin and the toxin: 1 to 3 hours, 24 hours, 7 days, 14more » days, and 90 days. It was found that, of the irradiated rabbits, 55% survived the toxin when the antitoxin was injected 1 to 3 hours after the exposure, 100% survived when the antitoxin was injected 24 hours after, 47% survived when the antitoxin was injected 7 days after, 27% survived when the antitoxin was injected 14 days after and none of the rabbits survived when the antitoxin was injected 90 days after. Of the non-irradiated controls, an average of 20% survived the toxin. The results indicated that a restricted amount of antitoxin injected subcutaneously in an irradiated area up to 7 days after exposure to 1000 r was more effective in the neutralization of a lethal dose of toxin than the same amount injected into a corresponding area of non-irradiated rabbits and the most marked neutralization occurred 24 hours after the exposure. By 14 days after the exposure, the neutralization of the toxin reached the control level, but 90 days after, no neutralization of the toxin was observed. The basis for the non-neutralization of the toxin in any of the rabbits was apparently not an insufficiency of the subcutaneouslyinjected antitoxin, which consisted of 15 units, since 5 units injected intravenously were found to be ample for neutralization of the same dose of toxin. The non-protection was due most likely to the localization of the proteins of the antitoxin, consisting of 0.75 mg, in the area of injection, preventing thereby the antitoxic fraction from reaching the toxin in time for neutralization. When the 15 units of antitoxin were injected in the irradiated area 1 to 3 hours after exposure to 1000 r, 45% of the rabbits showed localization of the protein. None of the rabbits showed localization when injected 24 hours after, 53% showed localization when injected 7 days after, 73% showed localization when injected 14 days after and 100% showed localization of the 0.75 mg protein when injected 90 days after the exposure to 1000 r. With regard to the controls, an average of 80% of the non-irradiated rabbits localized this fraction of a milligram of protein in the subcutaneously injected area. Localization in these experiments was undoubtedly temporary in nature, preceding proteolysis. But the length of time of localization was apparently sufficient to so retain the antitoxic fraction in the injected area as to prevent it from reaching and neutralizing the toxin. (auth)« less

9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

Code of Federal Regulations, 2014 CFR

2014-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Beta Antitoxin provided in this...

9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

Code of Federal Regulations, 2012 CFR

2012-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Beta Antitoxin provided in this...

9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

Code of Federal Regulations, 2014 CFR

2014-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Epsilon Antitoxin provided in this...

9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

Code of Federal Regulations, 2013 CFR

2013-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Epsilon Antitoxin provided in this...

9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

Code of Federal Regulations, 2013 CFR

2013-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Beta Antitoxin provided in this...

9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

Code of Federal Regulations, 2012 CFR

2012-01-01

... unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general requirements provided in § 113.450. (b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Epsilon Antitoxin provided in this...

Clostridium perfringens iota toxin: synergism between two proteins.

PubMed

Stiles, B G; Wilkins, T D

1986-01-01

The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme. Antiserum raised against C. spiroforme or C. perfringens type E neutralizes the toxin from both species. By using C. spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C. perfringens type E. Both proteins of C. perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone. However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively. These results demonstrate that the iota toxin of C. perfringens requires two immunologically and biochemically different proteins for maximum activity.

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

PubMed

Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

2015-11-13

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin’s Enzymatic Subunit Interfere with Intracellular Toxin Transport

PubMed Central

Yermakova, Anastasiya; Klokk, Tove Irene; O’Hara, Joanne M.; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J.

2016-01-01

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin’s enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin’s egress from EEA-1+ and Rab7+ vesicles and enhanced toxin accumulation in LAMP-1+ vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA. PMID:26949061

Structural and functional properties of antibodies to the superantigen TSST-1 and their relationship to menstrual toxic shock syndrome.

PubMed

Kansal, Rita; Davis, Catherine; Hansmann, Melanie; Seymour, Jon; Parsonnet, Jeffrey; Modern, Paul; Gilbert, Steve; Kotb, Malak

2007-05-01

Menstrual toxic shock syndrome (mTSS) is an acute febrile disease accompanied by hypotension and multiple organ involvement. Infection with Staphylococcus aureus producing the superantigen toxic shock syndrome toxin-1 (TSST-1) vaginally is necessary; however, only a small fraction of those infected with TSST-1 producing bacteria actually develop mTSS, suggesting that host factors modulate disease susceptibility. Serum antibodies to the toxin protect against development of the syndrome, but not all antibodies can neutralize the toxin. We set out to determine whether risk of developing the syndrome is related to the absence of neutralizing antibody and if antibody isotypes influence the neutralization capacity. In healthy subjects, TSST-1-binding serum antibodies were exclusively of the IgG and IgM classes; however, toxin-neutralizing capacity was correlated to the TSST-1-specific IgG1 and IgG4 antibodies (r (2)=0.88, p<0.0001 and 0.33, p<0.0086, respectively) but not with IgM antibodies. Specific IgA was not detectable. Compared to healthy matched controls who were colonized vaginally with S. aureus, IgG1 anti-TSST-1 antibodies and toxin neutralizing activity was lacking in all of the acute phases and in the majority of convalescent sera, suggesting that these patients may be incapable of generating TSST-1 neutralizing antibodies. These new findings support the hypothesis that host factors are important in the development of mTSS and that the anti-toxin isotype impacts antibody functionality.

Neutralization of B. anthracis toxins during ex vivo phagocytosis.

PubMed

Tarasenko, Olga; Scott, Ashley; Jones, April; Soderberg, Lee; Alusta, Pierre

2013-07-01

Glycoconjugates (GCs) are recognized as stimulation and signaling agents, affecting cell adhesion, activation, and growth of living organisms. Among GC targets, macrophages are considered ideal since they play a central role in inflammation and immune responses against foreign agents. In this context, we studied the effects of highly selective GCs in neutralizing toxin factors produced by B. anthracis during phagocytosis using murine macrophages. The effects of GCs were studied under three conditions: A) prior to, B) during, and C) following exposure of macrophages to B. anthracis individual toxin (protective antigen [PA], edema factor [EF], lethal factor [LF] or toxin complexes (PA-EF-LF, PA-EF, and PA-LF). We employed ex vivo phagocytosis and post-phagocytosis analysis including direct microscopic observation of macrophage viability, and macrophage activation. Our results demonstrated that macrophages are more prone to adhere to GC-altered PA-EF-LF, PA-EF, and PA-LF toxin complexes. This adhesion results in a higher phagocytosis rate and toxin complex neutralization during phagocytosis. In addition, GCs enhance macrophage viability, activate macrophages, and stimulate nitric oxide (NO) production. The present study may be helpful in identifying GC ligands with toxin-neutralizing and/or immunomodulating properties. In addition, our study could suggest GCs as new targets for existing vaccines and the prospective development of vaccines and immunomodulators used to combat the effects of B. anthracis.

Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

PubMed

Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

2016-08-01

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

PubMed Central

Ngundi, Miriam M.; Meade, Bruce D.; Little, Stephen F.; Quinn, Conrad P.; Corbett, Cindi R.; Brady, Rebecca A.

2012-01-01

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

PubMed

Souza, A M; Reis, J K P; Assis, R A; Horta, C C; Siqueira, F F; Facchin, S; Alvarenga, E R; Castro, C S; Salvarani, F M; Silva, R O S; Pires, P S; Contigli, C; Lobato, F C F; Kalapothakis, E

2010-02-18

Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

Differential neutralizing activities of a single domain camelid antibody (VHH) specific for ricin toxin's binding subunit (RTB).

PubMed

Herrera, Cristina; Vance, David J; Eisele, Leslie E; Shoemaker, Charles B; Mantis, Nicholas J

2014-01-01

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

PubMed

Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

2016-01-06

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structural Insights into Antibody Sequestering and Neutralizing of Na+ Channel α-Type Modulator from Old World Scorpion Venom

PubMed Central

Fabrichny, Igor P.; Mondielli, Grégoire; Conrod, Sandrine; Martin-Eauclaire, Marie-France; Bourne, Yves; Marchot, Pascale

2012-01-01

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na+ channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy. PMID:22371498

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms.

PubMed

Jiménez-Vargas, J M; Quintero-Hernández, V; González-Morales, L; Ortiz, E; Possani, L D

2017-03-15

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD 50 ) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD 50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 μL of serum from any of the immunized rabbits were enough to neutralize three LD 50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis.

PubMed

Goossens, Evy; Verherstraeten, Stefanie; Valgaeren, Bonnie R; Pardon, Bart; Timbermont, Leen; Schauvliege, Stijn; Rodrigo-Mocholí, Diego; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet R; Van Immerseel, Filip

2016-06-13

Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

PubMed Central

Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

2016-01-01

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile.

PubMed

Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N; Klassen, John S; Ng, Kenneth K S

2014-01-24

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile*

PubMed Central

Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N.; Klassen, John S.; Ng, Kenneth K. S.

2014-01-01

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents. PMID:24311789

Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin’s Binding Subunit (RTB)

PubMed Central

Herrera, Cristina; Vance, David J.; Eisele, Leslie E.; Shoemaker, Charles B.; Mantis, Nicholas J.

2014-01-01

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or “nanobody”) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1∶10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined. PMID:24918772

Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique

2011-08-09

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complexmore » reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.« less

Enterotoxigenic Escherichia coli heat-stable toxin and heat-labile toxin toxoid fusion 3xSTaN12S-dmLT induces neutralizing anti-STa antibodies in subcutaneously immunized mice.

PubMed

Nandre, Rahul; Ruan, Xiaosai; Duan, Qiangde; Zhang, Weiping

2016-11-02

Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers' diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin which is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTa N12S -dmLT, which consists of a monomer LT toxoid (LT R192G/L211A ) and three copies of STa toxoid STa N12S , may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTa N12S -dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTa N12S -dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits.

PubMed Central

Borriello, S P; Carman, R J

1983-01-01

A helically coiled, anaerobic, gram-positive sporeforming bacillus, identified as Clostridium spiroforme, was isolated from the cecal contents of all of 27 rabbits with spontaneous diarrhea, at a mean concentration of 10(6.0) spores per g of material. All of these rabbits also had a toxin present in their cecal contents that was neutralized by anti-Clostridium perfringens type E iota toxin, but not by other clostridial antitoxins. In addition, four rabbits with clindamycin-associated colitis were positive for C. spiroforme at a mean concentration of 10(4.5). All of these animals also had iota-like toxin present. Iota-like toxin was not detected in the cecal contents of 72 healthy animals, although C. spiroforme was found in two of these animals at a mean concentration of 10(6.0). C. spiroforme was shown to produce a toxin in vitro that was lethal to mice and caused dermonecrosis in guinea pigs. In all cases, this toxin was neutralized by anti-C. perfringens type E iota toxin. Images PMID:6841578

Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits.

PubMed

Borriello, S P; Carman, R J

1983-03-01

A helically coiled, anaerobic, gram-positive sporeforming bacillus, identified as Clostridium spiroforme, was isolated from the cecal contents of all of 27 rabbits with spontaneous diarrhea, at a mean concentration of 10(6.0) spores per g of material. All of these rabbits also had a toxin present in their cecal contents that was neutralized by anti-Clostridium perfringens type E iota toxin, but not by other clostridial antitoxins. In addition, four rabbits with clindamycin-associated colitis were positive for C. spiroforme at a mean concentration of 10(4.5). All of these animals also had iota-like toxin present. Iota-like toxin was not detected in the cecal contents of 72 healthy animals, although C. spiroforme was found in two of these animals at a mean concentration of 10(6.0). C. spiroforme was shown to produce a toxin in vitro that was lethal to mice and caused dermonecrosis in guinea pigs. In all cases, this toxin was neutralized by anti-C. perfringens type E iota toxin.

Vaccination of rhesus macaques with the anthrax vaccine adsorbed vaccine produces a serum antibody response that effectively neutralizes receptor-bound protective antigen in vitro.

PubMed

Clement, Kristin H; Rudge, Thomas L; Mayfield, Heather J; Carlton, Lena A; Hester, Arelis; Niemuth, Nancy A; Sabourin, Carol L; Brys, April M; Quinn, Conrad P

2010-11-01

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

STUDIES ON THE RELATIONSHIP OF STREPTOCOCCUS HEMOLYTICUS TO THE RHEUMATIC PROCESS

PubMed Central

Coburn, Alvin F.; Pauli, Ruth H.

1932-01-01

To determine the nature of the organisms associated with outbreaks of rheumatism at The Pelham Home, in a large number of individuals at the Presbyterian Hospital Nurses' Training School and among rheumatic subjects in New York City under continuous clinical observation, studies of the throat flora have been conducted. Hemolytic streptococcus in most instances appeared in the pharynx from 1 to 5 weeks before the onset of the rheumatic attack. These organisms have been investigated with the usual types of bacteriological tests and, in addition, have been classified serologically according to Lancefield's technique. The results have demonstrated that the organisms were not of a single type, but fell into six antigenic groups. The majority of the freshly isolated strains tested were strong toxin producers. The organisms producing the strongest toxin were cultures from the patients who developed extremely intense rheumatism. About 70 per cent of these toxins were neutralized by a monovalent streptococcus antiserum. PMID:19870090

Structural Analysis of Single Domain Antibodies Bound to a Second Neutralizing Hot Spot on Ricin Toxin's Enzymatic Subunit*

PubMed Central

Rudolph, Michael J.; Cassidy, Michael S.; Rong, Yinghui

2017-01-01

Ricin toxin is a heterodimer consisting of RTA, a ribosome-inactivating protein, and RTB, a lectin that facilitates receptor-mediated uptake into mammalian cells. In previous studies, we demonstrated that toxin-neutralizing antibodies target four spatially distinct hot spots on RTA, which we refer to as epitope clusters I–IV. In this report, we identified and characterized three single domain camelid antibodies (VHH) against cluster II. One of these VHHs, V5E1, ranks as one of the most potent ricin-neutralizing antibodies described to date. We solved the X-ray crystal structures of each of the three VHHs (E1, V1C7, and V5E1) in complex with RTA. V5E1 buries a total of 1,133 Å2 of surface area on RTA and makes primary contacts with α-helix A (residues 18–32), α-helix F (182–194), as well as the F-G loop. V5E1, by virtue of complementarity determining region 3 (CDR3), may also engage with RTB and potentially interfere with the high affinity galactose-recognition element that plays a critical role in toxin attachment to cell surfaces and intracellular trafficking. The two other VHHs, E1 and V1C7, bind epitopes adjacent to V5E1 but display only weak toxin neutralizing activity, thereby providing structural insights into specific residues within cluster II that may be critical contact points for toxin inactivation. PMID:27903650

Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

PubMed Central

Alfa, Michelle J; Sepehri, Shadi

2013-01-01

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile. PMID:24421808

9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

Code of Federal Regulations, 2011 CFR

2011-01-01

... of Standard Toxin to contain 10 Lo doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml. (iii) Combine 1 International Unit of Standard Antitoxin with 10 Lo doses of... 10 Lo doses of diluted Standard Toxin. (v) Neutralize all toxin-antitoxin mixtures at room...

9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

Code of Federal Regulations, 2014 CFR

2014-01-01

... of Standard Toxin to contain 10 Lo doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml. (iii) Combine 1 International Unit of Standard Antitoxin with 10 Lo doses of... 10 Lo doses of diluted Standard Toxin. (v) Neutralize all toxin-antitoxin mixtures at room...

9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

Code of Federal Regulations, 2013 CFR

2013-01-01

... of Standard Toxin to contain 10 Lo doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml. (iii) Combine 1 International Unit of Standard Antitoxin with 10 Lo doses of... 10 Lo doses of diluted Standard Toxin. (v) Neutralize all toxin-antitoxin mixtures at room...

9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

Code of Federal Regulations, 2012 CFR

2012-01-01

... of Standard Toxin to contain 10 Lo doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml. (iii) Combine 1 International Unit of Standard Antitoxin with 10 Lo doses of... 10 Lo doses of diluted Standard Toxin. (v) Neutralize all toxin-antitoxin mixtures at room...

Structural analysis of nested neutralizing and non-neutralizing B cell epitopes on ricin toxin's enzymatic subunit.

PubMed

Rudolph, Michael J; Vance, David J; Cassidy, Michael S; Rong, Yinghui; Shoemaker, Charles B; Mantis, Nicholas J

2016-08-01

In this report, we describe the X-ray crystal structures of two single domain camelid antibodies (VH H), F5 and F8, each in complex with ricin toxin's enzymatic subunit (RTA). F5 has potent toxin-neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Å(2) in complex with RTA and made contact with three prominent secondary structural elements: α-helix B (Residues 98-106), β-strand h (Residues 113-117), and the C-terminus of α-helix D (Residues 154-156). F8 buried 1103 Å(2) in complex with RTA that was centered primarily on β-strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions CDRs were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β-strand within RTA's centrally located β-sheet. A comparison of the two structures reported here to several previously reported (RTA-VH H) structures identifies putative contact sites on RTA, particularly α-helix B, associated with potent toxin-neutralizing activity. This information has implications for rational design of RTA-based subunit vaccines for biodefense. Proteins 2016; 84:1162-1172. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

PubMed Central

Linden, Jennifer R.; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole

2015-01-01

ABSTRACT Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. PMID:26081637

Occurrence of avian botulism in Korea during the period from June to September 2012.

PubMed

Jang, Il; Kang, Min-Su; Kim, Hye-Ryoung; Oh, Jae-Young; Lee, Jae-Il; Lee, Hee-Soo; Kwon, Yong-Kuk

2014-12-01

Botulism is a paralytic disease caused by the botulinum neurotoxin produced by Clostridium botulinum. In the summer season in Korea, intensive outbreaks of avian botulism were reported in both poultry and wild birds, including five Korean native chicken farms (HanHyup NO.3), one pheasant (Phasianus colchicus karpowi) farm, and one community of spot-billed ducks (Anas poecilorhyncha). The affected domestic birds showed 24.5% to 58.3% mortality, with specific clinical signs including ataxia, limber neck, and diarrhea. To confirm the botulinum toxin, neutralization tests were performed on sera (four Korean native chicken farms and one pheasant farm) or culture supernatant (spot-billed ducks). Additionally, the contents of the cecum and liver from poultry presenting signs suggestive of botulism were inoculated to isolate the pathogen. The toxin genes were then detected by polymerase chain reaction (PCR). Through the neutralization tests, it was possible to diagnose the botulism and, except in the case of one Korean native chicken farm, to identify the type of pathogen. Using detection by PCR, except in two cases of the Korean native chicken farms, the botulinum toxin gene was found. Additionally, in four cases, it was possible to identify the C/D mosaic type using PCR. This paper reports the first occurrence of avian botulism in domestic birds and the first detection of botulism caused by this mosaic type in Korea.

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin's binding subunit.

PubMed

Rong, Yinghui; Van Slyke, Greta; Vance, David J; Westfall, Jennifer; Ehrbar, Dylan; Mantis, Nicholas J

2017-01-01

Ricin toxin's binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB's high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB's high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

PubMed

Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

2015-09-01

Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins. Copyright © 2015 Elsevier B.V. All rights reserved.

Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois volitans).

PubMed

Kiriake, Aya; Shiomi, Kazuo

2011-11-01

Lionfish, members of the genera Pterois, Parapterois and Dendrochirus, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. The lionfish toxins have been shown to cross-react with the stonefish toxins by neutralization tests using the commercial stonefish antivenom, although their chemical properties including structures have been little characterized. In this study, an antiserum against neoverrucotoxin, the stonefish Synanceia verrucosa toxin, was first raised in a guinea pig and used in immunoblotting and inhibition immunoblotting to confirm that two species of Pterois lionfish (P. antennata and P. volitans) contain a 75kDa protein (corresponding to the toxin subunit) cross-reacting with neoverrucotoxin. Then, the amino acid sequences of the P. antennata and P. volitans toxins were successfully determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Notably, either α-subunits (699 amino acid residues) or β-subunits (698 amino acid residues) of the P. antennata and P. volitans toxins share as high as 99% sequence identity with each other. Furthermore, both α- and β-subunits of the lionfish toxins exhibit high sequence identity (70-80% identity) with each other and also with the β-subunits of the stonefish toxins. As reported for the stonefish toxins, the lionfish toxins also contain a B30.2/SPRY domain (comprising nearly 200 amino acid residues) in the C-terminal region of each subunit. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structural Analysis of Single Domain Antibodies Bound to a Second Neutralizing Hot Spot on Ricin Toxin's Enzymatic Subunit.

PubMed

Rudolph, Michael J; Vance, David J; Cassidy, Michael S; Rong, Yinghui; Mantis, Nicholas J

2017-01-20

Ricin toxin is a heterodimer consisting of RTA, a ribosome-inactivating protein, and RTB, a lectin that facilitates receptor-mediated uptake into mammalian cells. In previous studies, we demonstrated that toxin-neutralizing antibodies target four spatially distinct hot spots on RTA, which we refer to as epitope clusters I-IV. In this report, we identified and characterized three single domain camelid antibodies (V H H) against cluster II. One of these V H Hs, V5E1, ranks as one of the most potent ricin-neutralizing antibodies described to date. We solved the X-ray crystal structures of each of the three V H Hs (E1, V1C7, and V5E1) in complex with RTA. V5E1 buries a total of 1,133 Å 2 of surface area on RTA and makes primary contacts with α-helix A (residues 18-32), α-helix F (182-194), as well as the F-G loop. V5E1, by virtue of complementarity determining region 3 (CDR3), may also engage with RTB and potentially interfere with the high affinity galactose-recognition element that plays a critical role in toxin attachment to cell surfaces and intracellular trafficking. The two other V H Hs, E1 and V1C7, bind epitopes adjacent to V5E1 but display only weak toxin neutralizing activity, thereby providing structural insights into specific residues within cluster II that may be critical contact points for toxin inactivation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding properties of Clostridium botulinum type C progenitor toxin to mucins.

PubMed

Nakamura, Toshio; Takada, Noriko; Tonozuka, Takashi; Sakano, Yoshiyuki; Oguma, Keiji; Nishikawa, Atsushi

2007-04-01

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

PubMed

Yadirgi, G; Stickings, P; Rajagopal, S; Liu, Y; Sesardic, D

2017-12-01

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

PubMed

Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

2003-09-01

Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

PubMed Central

Little, S F; Leppla, S H; Cora, E

1988-01-01

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity. Images PMID:3384478

Antiradiation Antitoxin IgG : Immunological neutralization of Radiation Toxins at Acute Radiation Syndromes.

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Slava

Introduction: High doses of radiation induce apoptotic necrosis of radio-sensitive cells. Mild doses of radiation induce apoptosis or controlled programmed death of radio-sensitive cells with-out development of inflammation and formation of Radiation Toxins. Cell apoptotic necrosis initiates Radiation Toxins (RT)formation. Radiation Toxins play an important role as a trig-ger mechanism for inflammation development and cell lysis. If an immunotherapy approach to treatment of the acute radiation syndromes (ARS) were to be developed, a consideration could be given to neutralization of radiation toxins (Specific Radiation Determinants-SRD) by specific antiradiation antibodies. Therapeutic neutralization effects of the blocking anti-radiation antibodies on the circulated RT had been studied. Radiation Toxins were isolated from the central lymph of irradiated animals with Cerebrovascular(Cv ARS),Cardiovascular (Cr ARS),Gastrointestinal(Gi ARS) and Haemopoietic (Hp ARS) forms of ARS. To accomplish this objective, irradiated animals were injected with a preparation of anti-radiation immunoglobulin G (IgG) obtained from hyperimmune donors. Radiation-induced toxins that we call Specific Radiation Determinants (SRD) possess toxic (neurotoxic, haemotoxic) characteristics as well as specific antigenic properties. Depending on direct physiochemical radiation damage, they can induce development of many of the pathological processes associated with ARS. We have tested several specific hyperimmune IgG preparations against these radiation toxins and ob-served that their toxic properties were neutralized by the specific antiradiation IgGs. Material and Methods: A scheme of experiments was following: 1.Isolation of radiation toxins (RT) from the central lymph of irradiated animals with different form of ARS. 2.Transformation of a toxic form of the RT to a toxoid form of the RT. 3.Immunization of radiation naive animals. Four groups of rabbits were inoculated with a toxoid form of SRD radiation toxins to induce hyperimmune serum: Group A -Toxoid form of CV ARS toxins ( SRD-1); Group B-Toxoid form of CR ARS (SRD-2)toxins ; Group C -Toxoid form of GI ARS (SRD-3); Group D -Toxoid form of HP ARS (SRD-4). After the hyperimmune serum was pooled from several animals, purified, and concentrated, the IgG fraction was separated. Enzyme-linked immunosorbent assays of the hyper-immune serum had revealed high titers of IgG with specific binding to radi-ation toxins. The antiradiation IgG preparation was injected into laboratory animals one hour before and three hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. Results: Animals that were inoculated with specific antiradiation antibodies before and after receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate within 30 days. Also, these animals inoculated with the Antiradiation Antitoxin had exhibited markedly reduced clinical symptoms of the ARS, even those ones that did not survive irradiation. Discussion: The results of our experiments have demonstrated that the rabbit hyperimmune IgG preparations directed against SRD toxins provide a significant protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of animals treated by the hyperimmune IgG antidote have varied in the different groups of ani-mals and different forms of the ARS. However, significant radioprotection was observed in each group treated with the IgGs. The specific antiradiation antidote IGg isolated from hyperim-mune serum of immunized horses is under study. The specific antiradiation antidote contains antibodies to neurotoxins -SAAN IgG includes 50% IgG to Cv ARS, 25% IgG to Cr ARS and 25 % IgG to Gi ARS. The other type of the Specific antiradiation antidote containes antibodies to hematotoxins -SAAH IgG -100%. A combined variant is under consideration.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

PubMed Central

Wang, Xianzhe; Maynard, Jennifer A.

2015-01-01

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

Antitoxin activity of aqueous extract of Cyclea peltata root against Naja naja venom

PubMed Central

Sivaraman, Thulasi; Sreedevi, N. S.; Meenatchisundaram, S.; Vadivelan, R.

2017-01-01

OBJECTIVES: Snakebites are a significant and severe global health problem. Till date, anti-snake venom serum is the only beneficial remedy existing on treating the snakebite victims. As antivenom was reported to induce early or late adverse reactions to human beings, snake venom neutralizing potential for Cyclea peltata root extract was tested for the present research by ex vivo and in vivo approaches on Naja naja toxin. MATERIALS AND METHODS: Ex vivo evaluation of venom toxicity and neutralization assays was carried out. The root extracts from C. peltata were used to evaluate the Ex vivo neutralization tests such as acetylcholinesterase, protease, direct hemolysis assay, phospholipase activity, and procoagulant activity. Gas chromatography-mass spectrometry (GC-MS) analysis from root extracts of C. peltata was done to investigate the bioactive compounds. RESULTS: The in vivo calculation of venom toxicity (LD50) of N. naja venom remained to be 0.301 μg. C. peltata root extracts were efficiently deactivated the venom lethality, and effective dose (ED50) remained to be 7.24 mg/3LD50 of N. naja venom. C. peltata root extract was found effective in counteracting all the lethal effects of venom. GC-MS analysis of the plant extract revealed the presence of antivenom compounds such as tetradecanoic and octadecadienoic acid which have neutralizing properties on N. naja venom. CONCLUSION: The result from the ex vivo and in vivo analysis indicates that C. peltata plant root extract possesses significant compounds such as tetradecanoic acid hexadecanoic acid, heptadecanoic acid, and octadecadienoic acid which can counteract the toxins present in N. naja. PMID:29326487

Antitoxin activity of aqueous extract of Cyclea peltata root against Naja naja venom.

PubMed

Sivaraman, Thulasi; Sreedevi, N S; Meenatchisundaram, S; Vadivelan, R

2017-01-01

Snakebites are a significant and severe global health problem. Till date, anti-snake venom serum is the only beneficial remedy existing on treating the snakebite victims. As antivenom was reported to induce early or late adverse reactions to human beings, snake venom neutralizing potential for Cyclea peltata root extract was tested for the present research by ex vivo and in vivo approaches on Naja naja toxin. Ex vivo evaluation of venom toxicity and neutralization assays was carried out. The root extracts from C. peltata were used to evaluate the Ex vivo neutralization tests such as acetylcholinesterase, protease, direct hemolysis assay, phospholipase activity, and procoagulant activity. Gas chromatography-mass spectrometry (GC-MS) analysis from root extracts of C. peltata was done to investigate the bioactive compounds. The in vivo calculation of venom toxicity (LD 50 ) of N. naja venom remained to be 0.301 μg. C. peltata root extracts were efficiently deactivated the venom lethality, and effective dose (ED 50 ) remained to be 7.24 mg/3LD 50 of N. naja venom. C. peltata root extract was found effective in counteracting all the lethal effects of venom. GC-MS analysis of the plant extract revealed the presence of antivenom compounds such as tetradecanoic and octadecadienoic acid which have neutralizing properties on N. naja venom. The result from the ex vivo and in vivo analysis indicates that C. peltata plant root extract possesses significant compounds such as tetradecanoic acid hexadecanoic acid, heptadecanoic acid, and octadecadienoic acid which can counteract the toxins present in N. naja .

Antibody-Mediated Inhibition of Ricin Toxin Retrograde Transport

PubMed Central

Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J.

2014-01-01

ABSTRACT Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin’s binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. PMID:24713323

Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type B isolates in the mouse intravenous injection model.

PubMed

Fernandez-Miyakawa, Mariano E; Fisher, Derek J; Poon, Rachael; Sayeed, Sameera; Adams, Vicki; Rood, Julian I; McClane, Bruce A; Uzal, Francisco A

2007-03-01

Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

HOST-PARASITE FACTORS IN GROUP A STREPTOCOCCAL INFECTIONS

PubMed Central

Watson, Dennis W.

1960-01-01

The factors present in streptococcal lesion extracts (SLE) which enhanced the lethal and tissue-damaging properties of Gram-negative bacterial endotoxins and streptolysin O were identified with the scarlet fever group of toxins. Toxic manifestations attributed to this group of toxins included lethality, cardiotoxic and other tissue damage, enhancement of toxicity, and pyrogenicity. Of these, the measurement of febrile response in American Dutch rabbits was the most useful parameter of toxicity. In rabbits, repeated daily intravenous injections of 0.125 Lf of a purified erythrogenic toxin immunizes specifically against the pyrogenic activity; this technique was used to type the toxins and to distinguish them from exogenous and endogenous pyrogens; non-specific pyrogens, such as streptococcal endotoxin, were not found in SLE. All types of the Lancefield Group A streptococci tested produced one or or more immunologically distinct toxins in vivo in contrast to Groups B and C which did not produce them; toxins A and B, previously distinguished by neutralization of rash-inducing activity in the skin, were produced in vivo. The A toxin was the most common, as indicated by its presence in extracts prepared with Types 28, 12, 17, and 10 (NY-5); B toxin was found in 10 (NY-5) and 19. A new toxin, designated C, was obtained from a Type 18. In American Dutch rabbits, purified toxin at a concentration of 15 Lf (900,000 STD) neither gave a Dick test nor prepared the skin for the local Shwartzman reaction; by this route, however, in contrast to classical endotoxins, they enhance the lethal and tissue-damaging properties of sublethal doses of these and other toxins. These properties of the immunologic distinct exotoxins as demonstrated in American Dutch rabbits suggest by analogy their importance in the pathogenesis of streptococcal disease in man. Evidence that might implicate them in sequelae, in addition to scarlet fever, is discussed. PMID:13783427

Toxic Shock Syndrome Toxin-1-Mediated Toxicity Inhibited by Neutralizing Antibodies Late in the Course of Continual in Vivo and in Vitro Exposure

PubMed Central

Stich, Norbert; Model, Nina; Samstag, Aysen; Gruener, Corina S.; Wolf, Hermann M.; Eibl, Martha M.

2014-01-01

Toxic shock syndrome (TSS) results from the host’s overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure. PMID:24887085

Toxic shock syndrome toxin-1-mediated toxicity inhibited by neutralizing antibodies late in the course of continual in vivo and in vitro exposure.

PubMed

Stich, Norbert; Model, Nina; Samstag, Aysen; Gruener, Corina S; Wolf, Hermann M; Eibl, Martha M

2014-05-30

Toxic shock syndrome (TSS) results from the host's overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure.

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

PubMed Central

Sevigny, Leila M.; Booth, Brian J.; Rowley, Kirk J.; Leav, Brett A.; Cheslock, Peter S.; Garrity, Kerry A.; Sloan, Susan E.; Thomas, William; Babcock, Gregory J.

2013-01-01

Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic. PMID:23940209

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

PubMed

Bazzoli, Andrea; Vance, David J; Rudolph, Michael J; Rong, Yinghui; Angalakurthi, Siva Krishna; Toth, Ronald T; Middaugh, C Russell; Volkin, David B; Weis, David D; Karanicolas, John; Mantis, Nicholas J

2017-11-01

In this report we investigated, within a group of closely related single domain camelid antibodies (V H Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like V H Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1 R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7 G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function. © 2017 Wiley Periodicals, Inc.

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants.

PubMed

Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

2016-03-23

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

PubMed Central

Pellett, Sabine; Tepp, William H.; Bradshaw, Marite; Kalb, Suzanne R.; Dykes, Janet K.; Lin, Guangyun; Nawrocki, Erin M.; Pier, Christina L.; Barr, John R.; Maslanka, Susan E.

2016-01-01

ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. PMID:27303710

Effect of Irradiation and Test System on Development of Tetanus Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benenson, A. S.; Shively, J. N.; Vivona, S.

1963-03-01

Whole-body irradiation of dogs immunized with an alum precipitated tetanus toxoid results in a delay in appearance of antitoxin of the first toxoid us administered after radiation. Dogs irradiated 30 days after a first dose of toxoid and receiving a booster injection 24 hours after irradiation presented a good antitoxin response when measured by the hemagglutination test. However, a poor antibody response was found if the antitoxin was measured by the toxin neutralization techniques.

Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

PubMed

Rosenfeld, Ronit; Alcalay, Ron; Mechaly, Adva; Lapidoth, Gideon; Epstein, Eyal; Kronman, Chanoch; J Fleishman, Sarel; Mazor, Ohad

2017-09-01

While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Clinical Study of New Tetravalent (Type A, B, E, and F) Botulinum Toxoid Vaccine Derived from M Toxin in Japan.

PubMed

Torii, Yasushi; Sugimoto, Nakaba; Kohda, Tomoko; Kozaki, Shunji; Morokuma, Kazunori; Horikawa, Yoshikane; Ginnaga, Akihiro; Yamamoto, Akihiko; Takahashi, Motohide

2017-07-24

Botulinum toxin is the most poisonous substance known, and is believed to be a highly lethal as a biological weapon; researchers of the toxin are exposed to this hazard. Botulinum toxoid vaccines have been produced and used in Japan. However, since clinical studies involving these vaccines were conducted before establishment of the Ethical Guidelines for Clinical Research in Japan, their immunogenicity and safety were not systematically assessed. In this study, we produced a new tetravalent (type A, B, E, and F) botulinum toxoid vaccine, the first ever to be derived from M toxin, and conducted quality control tests with reference to the Minimum Requirements in Japan for adsorbed tetanus toxoid vaccine. Subsequently, a clinical study using the new vaccine in 48 healthy adult volunteers was conducted according to the guidelines in Japan. No clinically serious adverse event was noted. Neutralizing antibody titers for each type of toxin in the participants' sera, 1 month after the 4th injection were more than 0.25 IU/mL, indicating sufficient protection. This study demonstrated that the vaccine has marked immunogenicity and is safe for use in humans.

Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B ▿ †

PubMed Central

Permpoonpattana, Patima; Hong, Huynh A.; Phetcharaburanin, Jutarop; Huang, Jen-Min; Cook, Jenny; Fairweather, Neil F.; Cutting, Simon M.

2011-01-01

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A. PMID:21482682

Immunotherapy of acute radiation syndromes with antiradiation gamma G globulin.

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Vecheslav; Casey, Rachael; Jones, Jeffrey; Kedar, Prasad

Introduction: If an immunotherapy treatment approach to treatment of acute radiation syndromes (ARS) were to be developed; consideration could be given to neutralization of radiation toxins (Specific Radiation Determinants- SRD) by specific antiradiation antibodies. To accomplish this objective, irradiated animals were injected with a preparation of antiradiation immunoglobulin G (IgG) obtained from hyperimmune donors. Radiation-indeced toxins that we call Specific Radiation Determinants (SRD) possess toxic (neurotoxic, haemotoxic and enterotoxic) characteristics as well as specific antigenic properties that combined with the direct physiochemical direct radiation damage, induce the development of many of the pathological processes associated with ARS. We tested several specific hyperimmune IgG preparations against these radiation toxins and observed that their toxic properties were neutralized by specific antiradiation IgGs. Material and Methods: Rabbits were inoculated with SRD radiation toxins to induce hyperimmune serum. The hyperimmune serum was pooled from several animals, purified, and concentrated. Enzyme-linked immunosorbent assays of the hyperimmune serum revealed high titers of IgG with specific binding to radiation toxins. The antiradiation IgG preparation was injected into laboratory animals one hour before and three hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. Results: Animals that were inoculated with specific antiradiation antibodies before receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate at 30 days, whereas all control animals expired by 30 days following exposure. These inoculated animals also exhibited markedly reduced clinical symptoms of ARS, even those that did not survive irradiation. Discussion: The results of our experiments demonstrate that rabbit hyperimmune serum directed against SRD toxins afford significant, albeit incomplete, protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of hyperimmune serum-treated animals varied in different groups of animals and different forms of ARS; however, significant radioprotection was observed in each group treated with IgGs activated against specific radiation toxins.

Evidence-based recommendations on the use of intravenous lipid emulsion therapy in poisoning.

PubMed

Gosselin, Sophie; Hoegberg, Lotte C G; Hoffman, Robert S; Graudins, Andis; Stork, Christine M; Thomas, Simon H L; Stellpflug, Samuel J; Hayes, Bryan D; Levine, Michael; Morris, Martin; Nesbitt-Miller, Andrea; Turgeon, Alexis F; Bailey, Benoit; Calello, Diane P; Chuang, Ryan; Bania, Theodore C; Mégarbane, Bruno; Bhalla, Ashish; Lavergne, Valéry

2016-12-01

Although intravenous lipid emulsion (ILE) was first used to treat life-threatening local anesthetic (LA) toxicity, its use has expanded to include both non-local anesthetic (non-LA) poisoning and less severe manifestations of toxicity. A collaborative workgroup appraised the literature and provides evidence-based recommendations for the use of ILE in poisoning. Following a systematic review of the literature, data were summarized in four publications: LA and non-LA poisoning efficacy, adverse effects, and analytical interferences. Twenty-two toxins or toxin categories and three clinical situations were selected for voting. Voting statements were proposed using a predetermined format. A two-round modified Delphi method was used to reach consensus on the voting statements. Disagreement was quantified using RAND/UCLA Appropriateness Method. For the management of cardiac arrest, we recommend using ILE with bupivacaine toxicity, while our recommendations are neutral regarding its use for all other toxins. For the management of life-threatening toxicity, (1) as first line therapy, we suggest not to use ILE with toxicity from amitriptyline, non-lipid soluble beta receptor antagonists, bupropion, calcium channel blockers, cocaine, diphenhydramine, lamotrigine, malathion but are neutral for other toxins, (2) as part of treatment modalities, we suggest using ILE in bupivacaine toxicity if other therapies fail, but are neutral for other toxins, (3) if other therapies fail, we recommend ILE for bupivacaine toxicity and we suggest using ILE for toxicity due to other LAs, amitriptyline, and bupropion, but our recommendations are neutral for all other toxins. In the treatment of non-life-threatening toxicity, recommendations are variable according to the balance of expected risks and benefits for each toxin. For LA-toxicity we suggest the use of Intralipid ® 20% as it is the formulation the most often reported. There is no evidence to support a recommendation for the best formulation of ILE for non-LAs. The voting panel is neutral regarding ILE dosing and infusion duration due to insufficient data for non-LAs. All recommendations were based on very low quality of evidence. Clinical recommendations regarding the use of ILE in poisoning were only possible in a small number of scenarios and were based mainly on very low quality of evidence, balance of expected risks and benefits, adverse effects, laboratory interferences as well as related costs and resources. The workgroup emphasizes that dose-finding and controlled studies reflecting human poisoning scenarios are required to advance knowledge of limitations, indications, adverse effects, effectiveness, and best regimen for ILE treatment.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

PubMed

Rausch, Dana; Ruan, Xiaosai; Nandre, Rahul; Duan, Qiangde; Hashish, Emad; Casey, Thomas A; Zhang, Weiping

2017-04-01

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STa P12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LT R192G ), two toxoid MEFAs, 'LT R192G -STb-Stx2e-STa P12F ' and 'LT R192G -STb-Stx2e-3xSTa P12F ' which carried three copies of STa P12F , were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LT R192G -STb-Stx2e-3xSTa P12F ' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model*

PubMed Central

Moayeri, Mahtab; Leysath, Clinton E.; Tremblay, Jacqueline M.; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H.; Shoemaker, Charles B.

2015-01-01

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from “pre-pore” to its SDS and heat-resistant “pore” conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. PMID:25564615

A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

PubMed

Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

2015-03-06

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Deeper than skin deep - The effect of botulinum toxin-A on emotion processing.

PubMed

Baumeister, J-C; Papa, G; Foroni, F

2016-08-01

The effect of facial botulinum Toxin-A (BTX) injections on the processing of emotional stimuli was investigated. The hypothesis, that BTX would interfere with processing of slightly emotional stimuli and less with very emotional or neutral stimuli, was largely confirmed. BTX-users rated slightly emotional sentences and facial expressions, but not very emotional or neutral ones, as less emotional after the treatment. Furthermore, they became slower at categorizing slightly emotional facial expressions under time pressure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antiradiation Vaccine: Immunological neutralization of Radiation Toxins at Acute Radiation Syndromes.

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Slava

Introduction: Current medical management of the Acute Radiation Syndromes (ARS) does not include immune prophylaxis based on the Antiradiation Vaccine. Existing principles for the treatment of acute radiation syndromes are based on the replacement and supportive therapy. Haemotopoietic cell transplantation is recomended as an important method of treatment of a Haemopoietic form of the ARS. Though in the different hospitals and institutions, 31 pa-tients with a haemopoietic form have previously undergone transplantation with stem cells, in all cases(100%) the transplantants were rejected. Lethality rate was 87%.(N.Daniak et al. 2005). A large amount of biological substances or antigens isolated from bacterias (flagellin and derivates), plants, different types of venom (honeybees, scorpions, snakes) have been studied. This biological active substances can produce a nonspecific stimulation of immune system of mammals and protect against of mild doses of irradiation. But their radioprotection efficacy against high doses of radiation were not sufficient. Relative radioprotection characteristics or adaptive properties of antioxidants were expressed only at mild doses of radiation. However antioxidants demonstrated a very low protective efficacy at high doses of radiation. Some ex-periments demonstrated even a harmful effect of antioxidants administered to animals that had severe forms of the ARS. Only Specific Radiation Toxins roused a specific antigenic stim-ulation of antibody synthesis. An active immunization by non-toxic doses of radiation toxins includes a complex of radiation toxins that we call the Specific Radiation Determinant (SRD). Immunization must be provided not less than 24 days before irradiation and it is effective up to three years and more. Active immunization by radiation toxins significantly reduces the mortality rate (100%) and improves survival rate up to 60% compare with the 0% sur-vival rate among the irradiated animals in control groups. Material and Methods: The SRD molecules were isolated from Lymphatic Systems of animals that were irradiated with high doses of irradiation and had a clinical and laboratory picture of the Cerebral Acute Radia-tion Syndrome, Cardiovascular Acute Radiation Syndrome, Gastrointestinal Acute Radiation Syndrome, and Hematological Acute Radiation Syndrome. Our classification of radiation tox-ins includes 4 major groups: 1.SRD-1, Cerebrovascular neurotoxic Radiation Toxins (CvARS); 2.SRD-2, Cardiovascular Radiation Toxins(CrARS); 3.SRD-3,Gastrointestinal neurotoxic Ra-diation Toxins (GiARS); 4.SRD-4, Hematopietic Radiation Toxins (HpARS). Radiation tox-ins possess both toxic and immunological properties. But mechanisms of immune-toxicity by which radiation toxins stimulate development of the ARS are poorly understood. We have studied lethal toxicity of radiation toxins and an ability of specific antibodies to neutralize toxic activity of radiation toxins by specific antibodies. Results: The Blocking Antiradiation Antibodies induce an immunologically specific effect and inhibiting effects on radiation induced neuro-toxicity, vascular-toxicity, gastrointestinal toxcity, hematopoietic toxicity. Antiradiation Antibodies prevent the radiation induced cytolysis of selected groups of cells that are sensitive to radiation. The Blocking Antiradiation Antibodies are immunologically specific and can be produced by immunization with the different radiation toxins isolated from irradiated mam-mals. We propose that Specific Antiradiation Antibodies targeted against the radiation induced Toxins. Specific Antiradiation Antibodies neutralize toxic properties of radiation toxins. Anti-radiation Antibodies in different phases of the Acute Radiation Syndromes can compete with cytotoxic lymphocytes and prevent cytolysis mediated by cytotoxic lymphocytes. Conclusions: Immunological inhibition of cytotoxic and neurotoxic properties of Specific Radiation Toxins are significant factors for improving results of Medical Management of severe forms of the ARS and will optimize results of traditional methods of therapy of the ARS. Immunological inhi-bition of Radiation Toxins must be a part of technical procedure before haemotopoietic stem cells transplantation. Positive therapeutic results of neutralization of SRD RT could make a procedure of haemopoietic stem cell transplantation unnecessary.

Protective Immunity to Ricin Toxin Conferred by Antibodies against the Toxin’s Binding Subunit (RTB)

PubMed Central

Yermakova, Anastasiya; Mantis, Nicholas J.

2011-01-01

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5xLD50s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG1 antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5xLD50 dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB’s carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin. PMID:21872634

Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins

PubMed Central

1984-01-01

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field. PMID:6096479

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants

PubMed Central

Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

2016-01-01

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals. PMID:27004612

DNA aptamers as a novel approach to neutralize Staphylococcus aureus α-toxin.

PubMed

Vivekananda, Jeevalatha; Salgado, Christi; Millenbaugh, Nancy J

2014-02-14

Staphylococcus aureus is a versatile pathogen capable of causing a broad spectrum of diseases ranging from superficial skin infections to life threatening conditions such as endocarditis, septicemia, pneumonia and toxic shock syndrome. In vitro and in vivo studies identified an exotoxin, α-toxin, as a major cause of S. aureus toxicity. Because S. aureus has rapidly evolved resistance to a number of antibiotics, including methicillin, it is important to identify new therapeutic strategies, other than antibiotics, for inhibiting the harmful effects of this pathogen. Aptamers are single-stranded DNA or RNA oligonucleotides with three-dimensional folded conformations that bind with high affinity and selectivity to targets and modulate their biological functions. The goal of this study was to isolate DNA aptamers that specifically inhibit the cytotoxic activity of α-toxin. After 10 rounds of Systematic Evolution of Ligands by EXponential Enrichment (SELEX), 49 potential anti-α-toxin aptamers were identified. In vitro neutralization assays demonstrated that 4 of these 49 aptamers, AT-27, AT-33, AT-36, and AT-49, significantly inhibited α-toxin-mediated cell death in Jurkat T cells. Furthermore, RT-PCR analysis revealed that α-toxin increased the transcription of the inflammatory cytokines TNF-α and IL-17 and that anti-α-toxin aptamers AT-33 and AT-36 inhibited the upregulation of these genes. Collectively, the data suggest the feasibility of generating functionally effective aptamers against α-toxin for treatment of S. aureus infections. Published by Elsevier Inc.

Identification of the factors that govern the ability of therapeutic antibodies to provide postchallenge protection against botulinum toxin: a model for assessing postchallenge efficacy of medical countermeasures against agents of bioterrorism and biological warfare.

PubMed

Al-Saleem, Fetweh H; Nasser, Zidoon; Olson, Rebecca M; Cao, Linsen; Simpson, Lance L

2011-08-01

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare.

Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar.

PubMed

Marston, Chung K; Ibrahim, Hisham; Lee, Philip; Churchwell, George; Gumke, Megan; Stanek, Danielle; Gee, Jay E; Boyer, Anne E; Gallegos-Candela, Maribel; Barr, John R; Li, Han; Boulay, Darbi; Cronin, Li; Quinn, Conrad P; Hoffmaster, Alex R

2016-01-01

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.

Neutralizing antibodies to botulinum neurotoxin type A in aesthetic medicine: five case reports

PubMed Central

Torres, Sebastian; Hamilton, Mark; Sanches, Elena; Starovatova, Polina; Gubanova, Elena; Reshetnikova, Tatiana

2014-01-01

Botulinum neurotoxin injections are a valuable treatment modality for many therapeutic indications as well as in the aesthetic field for facial rejuvenation. As successful treatment requires repeated injections over a long period of time, secondary resistance to botulinum toxin preparations after repeated injections is an ongoing concern. We report five case studies in which neutralizing antibodies to botulinum toxin type A developed after injection for aesthetic use and resulted in secondary treatment failure. These results add to the growing number of reports in the literature for secondary treatment failure associated with high titers of neutralizing antibodies in the aesthetic field. Clinicians should be aware of this risk and implement injection protocols that minimize resistance development. PMID:24379687

Disease-enhancing antibodies improve the efficacy of bacterial toxin-neutralizing antibodies

PubMed Central

Chow, Siu-Kei; Smith, Cameron; MacCarthy, Thomas; Pohl, Mary Ann; Bergman, Aviv; Casadevall, Arturo

2013-01-01

SUMMARY During infection, humoral immunity produces a polyclonal response with various immunoglobulins recognizing different epitopes within the microbe or toxin. Despite this diverse response, the biological activity of an antibody (Ab) is usually assessed by the action of a monoclonal population. We demonstrate that a combination of monoclonal antibodies (mAbs) that are individually disease-enhancing or neutralizing to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, results in significantly augmented protection against the toxin. This boosted protection is Fc gamma receptor (FcγR)-dependent and involves the formation of stoichiometrically defined mAb-PA complexes that requires immunoglobulin bivalence and simultaneous interaction between PA and the two mAbs. The formation of these mAb-PA complexes inhibits PA oligomerization, resulting in protection. These data suggest that functional assessments of single Abs may inaccurately predict how the same Abs will operate in polyclonal preparations and imply that potentially therapeutic mAbs may be overlooked in single Ab screens. PMID:23601104

Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs

PubMed Central

Diamant, Eran; Torgeman, Amram; Ozeri, Eyal; Zichel, Ran

2015-01-01

Monoclonal antibodies (MAbs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed. PMID:26035486

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model.

PubMed

Ndumnego, Okechukwu C; Köhler, Susanne M; Crafford, Jannie; van Heerden, Henriette; Beyer, Wolfgang

2016-10-01

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination. Copyright © 2016 Elsevier B.V. All rights reserved.

Neutralizing activity and protective immunity to ricin toxin conferred by B subunit (RTB)-specific Fab fragments.

PubMed

Yermakova, Anastasiya; Mantis, Nicholas J

2013-09-01

SylH3 and 24B11 are murine monoclonal antibodies directed against different epitopes on ricin toxin's binding (RTB) subunit that have been shown to passively protect mice against ricin challenge. Here we report that Fab fragments of SylH3 and 24B11 neutralize ricin in a cell based assay, and in a mouse challenge model as effectively as their respective full length parental IgGs. These data demonstrate that immunity to ricin can occur independent of Fc-mediated clearance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human immune response to botulinum pentavalent (ABCDE) toxoid determined by a neutralization test and by an enzyme-linked immunosorbent assay.

PubMed Central

Siegel, L S

1988-01-01

To determine the immune status of persons receiving botulinum pentavalent (ABCDE) toxoid and to evaluate the effectiveness of the vaccine, we surveyed immunized individuals for neutralizing antibodies to type A and to type B botulinum toxins. After the primary series of three immunizations administered at 0, 2, and 12 weeks, 21 of 23 persons tested (91%) had a titer for type A that was greater than or equal to 0.08 international units (IU)/ml, and 18 (78%) had a titer for type B of greater than or equal to 0.02 IU/ml. (One international unit is defined as the amount of antibody neutralizing 10,000 mouse 50% lethal doses of type A or B botulinum toxin). Just before the first annual booster, 10 of 21 (48%) and 14 of 21 (67%) people lacked a detectable titer for type A and for type B, respectively. After the first booster, all individuals tested had a demonstrable titer to both types A and B. Of 77 persons who had previously received from one to eight boosts of the toxoid, 74 (96%) had an A titer of greater than or equal to 0.25 IU/ml and would not require an additional booster, according to the recommendations of the Centers for disease Control. However, only 44 of 77 (57%) had a B titer of greater than or equal to 0.25 IU/ml. In each group by booster number, even the group having had eight boosts, at least one person would require reimmunization on the basis of B titer. There was a wide range of antibody levels among individuals at the same point in the immunization scheme. Results from an enzyme linked immunosorbent assay, with purified type A or type B neurotoxin as the capture antigen, were compared with neutralization test results on 186 serum samples for type A and 168 samples for type B. Statistically, the correlation coefficients for results from the two assays were high (r = 0.69, P < 0.0001, for type A and r = 0.77, P < 0.0001, for type B). However, due to the wide dispersion of values obtained, using enzyme-linked immunosorbent assay results to predict neutralizing antibody levels is unwarranted. PMID:3235662

Identification of the Factors That Govern the Ability of Therapeutic Antibodies to Provide Postchallenge Protection Against Botulinum Toxin: A Model for Assessing Postchallenge Efficacy of Medical Countermeasures against Agents of Bioterrorism and Biological Warfare

PubMed Central

Al-Saleem, Fetweh H.; Nasser, Zidoon; Olson, Rebecca M.; Cao, Linsen

2011-01-01

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare. PMID:21586604

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models.

PubMed

Tian, Jing-Hui; Glenn, Gregory; Flyer, David; Zhou, Bin; Liu, Ye; Sullivan, Eddie; Wu, Hua; Cummings, James F; Elllingsworth, Larry; Smith, Gale

2017-07-24

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB (027) ). TcdB (027) is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB (003) ). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB (003) , TcdB (027) and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB (003) /TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB (003) /TcdA/TcdB (027) ) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

PubMed

Brown, J E; Rothman, S W; Doctor, B P

1980-07-01

Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and deoxyribonucleic acid syntheses. The inhibitory effect of toxin was sensitive to heat inactivation and was prevented by antibody neutralization. Several cytotoxic components were separated by polyacrylamide gel electrophoresis of the purified toxin preparations; all inhibited protein and deoxyribonucleic acid syntheses equally.

Polyvalent Recognition of Biopolymers:The Design of Potent Inhibitors of Anthrax Toxin

NASA Astrophysics Data System (ADS)

Kane, Ravi

2007-03-01

Polyvalency -- the simultaneous binding of multiple ligands on one entity to multiple receptors on another -- is a phenomenon that is ubiquitous in nature. We are using a biomimetic approach, inspired by polyvalency, to design potent inhibitors of anthrax toxin. Since the major symptoms and death from anthrax are due primarily to the action of anthrax toxin, the toxin is a prime target for therapeutic intervention. We describe the design of potent polyvalent anthrax toxin inhibitors, and will discuss the role of pattern matching in polyvalent recognition. Pattern-matched polyvalent inhibitors can neutralize anthrax toxin in vivo, and may enable the successful treatment of anthrax during the later stages of the disease, when antibiotic treatment is ineffective.

Toxin-Based Therapeutic Approaches

PubMed Central

Shapira, Assaf; Benhar, Itai

2010-01-01

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin. PMID:22069564

Characterization of Heat-Stable (STa) Toxoids of Enterotoxigenic Escherichia coli Fused to Double Mutant Heat-Labile Toxin Peptide in Inducing Neutralizing Anti-STa Antibodies

PubMed Central

Ruan, Xiaosai; Robertson, Donald C.; Nataro, James P.; Clements, John D.

2014-01-01

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The results showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced by the 3×STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested 3×STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited information for effective ETEC vaccine development. PMID:24549325

Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination.

PubMed

Linden, Jennifer R; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole; Vartanian, Timothy

2015-06-16

Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. Our intestinal tract is host to trillions of microorganisms that play an essential role in health and homeostasis. Disruption of this symbiotic relationship has been implicated in influencing or causing disease in distant organ systems such as the brain. Epsilon toxin (ε-toxin)-carrying Clostridium perfringens strains are responsible for a devastating white matter disease in ruminant animals that shares similar features with human multiple sclerosis. In this report, we define the mechanism by which ε-toxin causes white matter disease. We find that ε-toxin specifically targets the myelin-forming cells of the central nervous system (CNS), oligodendrocytes, leading to cell death. The selectivity of ε-toxin for oligodendrocytes is remarkable, as other cells of the CNS are unaffected. Importantly, ε-toxin-induced oligodendrocyte death results in demyelination and is dependent on expression of myelin and lymphocyte protein (MAL). These results help complete the mechanistic pathway from bacteria to brain by explaining the specific cellular target of ε-toxin within the CNS. Copyright © 2015 Linden et al.

Nanoparticle-detained toxins for safe and effective vaccination

NASA Astrophysics Data System (ADS)

Hu, Che-Ming J.; Fang, Ronnie H.; Luk, Brian T.; Zhang, Liangfang

2013-12-01

Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

Clostridium perfringens Iota-Toxin: Mapping of Receptor Binding and Ia Docking Domains on Ib

PubMed Central

Marvaud, Jean-Christophe; Smith, Theresa; Hale, Martha L.; Popoff, Michel R.; Smith, Leonard A.; Stiles, Bradley G.

2001-01-01

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing ≥200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice. PMID:11254604

Improved Formulation of a Recombinant Ricin A-chain Vaccine Increases its Stability and Effective Antigenicity

DTIC Science & Technology

2007-03-26

Adjuvant adsorption; Toxin . Introduction Ricin, a highly potent toxin derived from the castor bean, ills human cells by depurinating a specific...moieties n the cell surface. Antibodies elicited against either the ricin (RTA) or B-chain can neutralize the toxin, although anti- Abbreviations: RTA...human B- cell epitope for RTA has been identified by astelletti et al. [6] from cancer patients treated with a ricin- onjugate immunotoxin, and lies

[Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice].

PubMed

Wang, Xiaolin; Chi, Xiangyang; Liu, Ju; Liu, Weicen; Liu, Shuling; Qiu, Shunfang; Wen, Zhonghua; Fan, Pengfei; Liu, Kun; Song, Xiaohong; Fu, Ling; Zhang, Jun; Yu, Changming

2016-11-25

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin.

PubMed

Sully, Erin K; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W; Stavale, Eric; Aman, M Javad; Tangudu, Chandra; Uzal, Francisco A; Mantis, Nicholas J; Zeitlin, Larry

2014-12-15

Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies

USDA-ARS?s Scientific Manuscript database

Escherichia coli (E. coli) food contaminations pose serious health and food safety concerns, and have been the subject of massive food recalls. Shiga toxin 2 (Stx2)-producing E. coli has been identified as the major cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS), the most severe di...

Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karalewitz, Andrew P.-A.; Kroken, Abby R.; Fu, Zhuji

2010-09-22

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical andmore » cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.« less

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

DOE PAGES

Dover, Nir; Barash, Jason R.; Burke, Julianne N.; ...

2014-05-22

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bontmore » gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ 70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.« less

Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine.

PubMed

McComb, Ryan C; Martchenko, Mikhail

2016-01-02

Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed (AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a well-defined formulation but these vaccines have been shown to lose potency as they are stored. In addition, studies have shown that a proportion of the antibody response against these vaccines is focused on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses on designing vaccine antigens based on structural information provided by neutralizing antibody epitope mapping, crystal structure analysis, and functional mapping through amino acid mutations. This information provides an opportunity to design antigens that target only functionally important and conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides an overview of the literature related to functional and neutralizing antibody epitope mapping of the Protective Antigen (PA) component of anthrax toxin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

PubMed Central

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-01-01

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

PubMed

Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

2016-03-03

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

[Morphologic changes in cultures of different tissues exposed to the toxins of C1. perfringens types B, C, E and F].

PubMed

Ermakova, M P; Zemlianitskaia, E P

1975-11-01

There were revealed morphological peculiarities of the action of C1. perfringens toxins, types B, C, D, E and F on the cultures of fibroblasts of chick embryo, amniotic cells and intestinal tissue. The toxin type B was characterized by a marked vocuolization of the cell cytoplasm; the action of the toxin of type C was expressed in the swelling of the nuclei and the lysis of the chromatine substance, the toxin of type E casued kariorhexis, and the toxin of type F--hyperchromatosis of the nuclei. All the cultures proved to be insensitive to the toxin of type D. Peculiarity of the morphological affection of the cells permitted to differentiate toxin of type B in the cultures of the fibroblasts of chick embryo, whereas the toxins of types C, E and F--in the cultures of the amniotic cells under control of the reaction of neutralization with the homologous antitoxic sera.

Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity.

PubMed

Pittman, Phillip R; Fisher, Diana; Quinn, Xiaofei; Schmader, Trevor; Barrera-Oro, Julio G

2013-10-17

We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax(®)) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18-24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P<0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66 μg/mL before and 887.82 μg/mL after the boost (P<0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series. Published by Elsevier Ltd.

Variability of Venom-Neutralizing Properties of Serum from Snakes of the Colubrid Genus Lampropeltis

DTIC Science & Technology

1992-01-01

venoms of C. atrx , S. m. bar- potentials for C s. scauhatus (type B) venom bouri, or A. c. mokasen showed persistent (Table 2). inflammation and/or edema...SMITH propeltis with these toxins would cause vascu- thality. Other workers have proposed antibody lotoxic effects. Klauber (1956) reported immu...tested, those injected with venom alone. This suggests Harvey (1960) described inhibition of C. atrx that elapid venom myolytic phospholipases Al venom

Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish.

PubMed

Erdenlig, S; Ainsworth, A J; Austin, F W

1999-07-01

We produced monoclonal antibodies (MAbs) to the extracellular proteins of Listeria monocytogenes EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from Listeria ivanovii. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of L. monocytogenes. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from L. ivanovii and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between L. monocytogenes PC-PLC and L. ivanovii sphingomyelinase. The reactions of the MAbs with catfish isolates of L. monocytogenes suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of L. monocytogenes in food and clinical samples and for detecting L. ivanovii in veterinary clinical samples.

Comparison of Simplexa universal direct PCR with cytotoxicity assay for diagnosis of Clostridium difficile infection: performance, cost, and correlation with disease.

PubMed

Landry, Marie L; Ferguson, David; Topal, Jeffrey

2014-01-01

Simplexa Clostridium difficile universal direct PCR, a real-time PCR assay for the detection of the C. difficile toxin B (tcdB) gene using the 3M integrated cycler, was compared with a two-step algorithm which includes the C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Three hundred forty-two liquid or semisolid stools submitted for diagnostic C. difficile testing, 171 GDH antigen positive and 171 GDH antigen negative, were selected for the study. All samples were tested by the C. Diff Chek-60 GDH antigen assay, cytotoxin neutralization, and Simplexa direct PCR. Of 171 GDH-positive samples, 4 were excluded (from patients on therapy or from whom duplicate samples were obtained) and 88 were determined to be true positives for toxigenic C. difficile. Of the 88, 67 (76.1%) were positive by the two-step method and 86 (97.7%) were positive by PCR. Seventy-nine were positive by the GDH antigen assay only. Of 171 GDH antigen-negative samples, none were positive by PCR. One antigen-negative sample positive by the cytotoxin assay only was deemed a false positive based on chart review. Simplexa C. difficile universal direct PCR was significantly more sensitive for detecting toxigenic C. difficile bacteria than cytotoxin neutralization (P = 0.0002). However, most PCR-positive/cytotoxin-negative patients did not have clear C. difficile disease. The estimated cost avoidance provided by a more rapid molecular diagnosis was outweighed by the cost of isolating and treating PCR-positive/cytotoxin-negative patients. The costs, clinical consequences, and impact on nosocomial transmission of treating and/or isolating patients positive for toxigenic C. difficile by PCR but negative for in vivo toxin production merit further study.

The CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Batot, Gaëlle; Michalska, Karolina; Ekberg, Greg

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(Ykris) complex from Yersinia kris-tensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistentmore » with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiI(Ykris) binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase Alike toxins are commonly deployed in inter-bacterial competition.« less

Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats.

PubMed

Ndumnego, Okechukwu C; Crafford, Jannie; Beyer, Wolfgang; van Heerden, Henriette

2013-12-27

Presently, few data exist on the level and duration of anti-protective antigen (PA) IgG in vaccinated livestock. Various adaptation of enzyme-linked immunosorbent assays (ELISAs) have been developed in studies to assess immune response following vaccination, albeit mostly in laboratory rodent models. The quantitative anti-anthrax IgG ELISA in this study describes a method of enumerating the concentration of anti-PA specific IgG present in sera of immunized goats, with the aid of an affinity-purified caprine polyclonal anti-anthrax PA-83 IgG standard. This was compared with the anthrax toxin neutralization assay (TNA) which measures a functional subset of toxin neutralizing anti-PA IgG. The measured concentrations obtained in the standard curve correlated with the known concentration at each dilution. Percentage recovery of the standard concentrations ranged from 89 to 98% (lower and upper asymptote respectively). Mean correlation coefficient (r2) of the standard curve was 0.998. Evaluation of the intra-assay coefficient of variation showed ranges of 0.23-16.90% and 0.40-12.46% for days 28 and 140 sera samples respectively, following vaccination. The mean inter-assay coefficient of variation for triplicate samples repeated on 5 different days was 18.53 and 12.17% for days 28 and 140 sera samples respectively. Spearman's rank correlation of log-transformed IgG concentrations and TNA titres showed strong positive correlation (rs = 0.942; p = 0.01). This study provides evidence that an indirect ELISA can be used for the quantification of anti-anthrax PA IgG in goats with the added advantage of using single dilutions to save time and resources. The use of such related immunoassays can serve as potential adjuncts to potency tests for Sterne and other vaccine types under development in ruminant species. This is the first report on the correlation of polyclonal anti-anthrax PA83 antibody with the TNA in goats.

Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens.

PubMed

Vance, David J; Greene, Christopher J; Rong, Yinghui; Mandell, Lorrie M; Connell, Terry D; Mantis, Nicholas J

2015-12-01

Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 μg when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa's activity in humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

PubMed

Prezoto, B C; Tanaka-Azevedo, A M; Marcelino, J R; Tashima, A K; Nishiduka, E S; Kapronezai, J; Mota, J O; Rocha, M M T; Serino-Silva, C; Oguiura, N

2018-06-15

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control. Copyright © 2018 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Johnson, Parker M.; Stols, Lucy

Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI +bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein fromNeisseria meningitidisMC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of RNA-binding proteins, but appears to lack the characteristic nucleic acid-binding motif of this family. Sequence homologymore » suggests that the cognate CdiA-CT is related to the eukaryotic EndoU family of RNA-processing enzymes. A homology model is presented of the CdiA-CT based on the structure of the XendoU nuclease fromXenopus laevis. Molecular-docking simulations predict that the CdiA-CT toxin active site is occluded upon binding to the CdiI immunity protein. Together, these observations suggest that the immunity protein neutralizes toxin activity by preventing access to RNA substrates.« less

THE PRODUCTION OF DIPHTHERIA TOXIN

PubMed Central

Park, W. H.; Williams, A. W.

1896-01-01

Toxin of sufficient strength to kill a 400-gramme guinea-pig in three days and a half in a dose of 0·cubic centimetre developed in suitable bouillon, contained in ordinary Erlenmeyer flasks, within a period of twenty-four hours. In such boullon the toxin reached its greatest strength in from four to seven days (0·005 cubic centimetre killing a 500-gramme guinea-pig in three days). This period of time covered that of the greatest growth of the bacilli, as shown both by the appearance of the culture and by the number of colonies developing an agar plates. The bodies of the diphtheria bacili did not at any time contain toxin in cosiderable amounts. The type of growth of the bacili and the rapidity and extent of the production of toxin depended more on the reaction of the bouillon than upon any other single factor. The best results were obtained in bouillon which, after being neutralized to litmus, had about seven cubic centimetres of normal soda solution added to each litre. An excessive amount of either acid or alkali prevented the development of toxin. Strong toxin was produced in bouillon containing peptone ranging from one to ten per cent. The strength of toxin averaged greater in the two and four-per-cent peptone solutions than in the one-percent. When the stage of acid reaction was brief and the degree of acidity probably slight, strong toxin developed while the culture bouillon was still acid; but when the stage of acid reaction was prolonged, little if any toxin was produced until just before the fluid became alkaline. Glucose is deleterious to the growth of the diphtheria bacillus and to the production of toxin when it is present in sufficient amounts to cause by its disintegration too great a degree of acidity in the fluid culture. When the acid resulting from decomposition of glucose is neutralized by the addition of alkali the diphtheria bacilus again grows abundantly. Glucose is not present, at least as a rule, in sufficient amounts in the meat as obtained from the New York butchers to prevent the rapid production of strong toxin if the bouillon is made sufficiently alkaline. In our experiments, when other conditions were similar, the strength of the toxin was in proportion to the virulence and vigour of growth of the bacillus employed. PMID:19866791

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

PubMed

Monti, Maria C; Hernández-Arriaga, Ana M; Kamphuis, Monique B; López-Villarejo, Juan; Heck, Albert J R; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H H

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

PubMed

Jolivet-Reynaud, C; Hauttecoeur, B; Alouf, J E

1989-01-01

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Role of Fc in Antibody-Mediated Protection from Ricin Toxin

PubMed Central

Pincus, Seth. H.; Das, Anushka; Song, Kejing; Maresh, Grace A.; Corti, Miriam; Berry, Jody

2014-01-01

We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab’)2 preparations should be balanced against possible loss of protective efficacy. PMID:24811206

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

PubMed

Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

2015-12-02

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. Copyright © 2015, American Association for the Advancement of Science.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

PubMed Central

Nguyen, Annalee W.; Wagner, Ellen K.; Laber, Joshua R.; Goodfield, Laura L.; Smallridge, William E.; Harvill, Eric T.; Papin, James F.; Wolf, Roman F.; Padlan, Eduardo A.; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A.

2016-01-01

In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. PMID:26631634

Antibody response to the epsilon toxin of Clostridium perfringens following vaccination of Lama glama crias.

PubMed

Bentancor, Adriana B; Halperin, Pablo; Flores, Myriam; Iribarren, Fabián

2009-09-15

Enterotoxaemia produced by Clostridium perfringens A, C and D is an important cause of mortality in young llamas. There is no data on antibody responses following vaccination with epsilon toxin. Twenty-six L. glama crias were divided into four groups which were vaccinated with a commercial vaccine (Mancha Gangrena Enterotoxemia, Instituto Rosembusch Sociedad Anónima, Argentina) on days 0, 21 and 42 or left as unvaccinated controls. An indirect ELISA was compared with the mouse neutralization test (MNT) for measuring titers to C. perfringens type D epsilon toxin and used to determine titers in sera taken before vaccination and 16, 28, 49, 59, and 93 days later. The ELISA gave comparable results to the MNT and showed animals vaccinated once failed to develop raised titers. A week following a second vaccination, mean antibody titers rose significantly (P < 0.05) and 7/12 animals developed high titers which were present in only one animal at the end of the study (day 93). A third vaccination resulted in a decrease in mean antibody titers a week later. Llamas develop antibodies to Clostridium perfringens type D epsilon toxin after two vaccinations at a 21-day interval. Further studies are indicated to determine if these inoculations protect against enterotoxemia and the most appropriate vaccination schedule.

Assembly dynamics and stability of the pneumococcal epsilon zeta antitoxin toxin (PezAT) system from Streptococcus pneumoniae.

PubMed

Mutschler, Hannes; Reinstein, Jochen; Meinhart, Anton

2010-07-09

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.

Cobra venom cytotoxins; apoptotic or necrotic agents?

PubMed

Ebrahim, Karim; Shirazi, Farshad H; Mirakabadi, Abbas Zare; Vatanpour, Hossein

2015-12-15

Organs homeostasis is controlled by a dynamic balance between cell proliferation and apoptosis. Failure to induction of apoptosis has been implicated in tumor development. Cytotoxin-I (CTX-I) and cytotoxin-II (CTX-II) are two physiologically active polypeptides found in Caspian cobra venom. Anticancer activity and mechanism of cell death induced by these toxins have been studied. The toxins were purified by different chromatographic steps and their cytotoxicity and pattern of cell death were determined by MTT, LDH release, acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis, caspase-3 activity and neutral red assays. The IC50 of CTX-II in MCF-7, HepG2, DU-145 and HL-60 was 4.1 ± 1.3, 21.2 ± 4.4, 9.4 ± 1.8 μg/mL and 16.3 ± 1.9 respectively while the IC50 of this toxin in normal MDCK cell line was 54.5 ± 3.9 μg/mL. LDH release suddenly increase after a specific toxins concentrations in all cell lines. AO/EtBr double staining, flow cytometric analysis and caspase-3 activity assay confirm dose and time-dependent induction of apoptosis by both toxins. CTX-I and CTX-II treated cells lost their lysosomal membrane integrity and couldn't uptake neutral red day. CTX-I and CTX-II showed significant anticancer activity with minimum effects on normal cells and better IC50 compared to current anticancer drug; cisplatin. They induce their apoptotic effect via lysosomal pathways and release of cathepsins to cytosol. These effects were seen in limited rage of toxins concentrations and pattern of cell death rapidly changes to necrosis by increase in toxin's concentration. In conclusion, significant apoptogenic effects of these toxins candidate them as a possible anticancer agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia.

PubMed

Das, Shreya; Majumder, Saugata; Kingston, Joseph J; Batra, Harsh V

2016-02-01

Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the most lethal forms of haemorrhagic and necrotic enteritis and sudden death syndrome affecting especially neonates. While CPB enterotoxemia is one of the most common forms of clostridial enterotoxemia, CPI enterotoxemia though putatively considered to be rare is an emerging cause of concern. The similarities in clinical manifestation, gross and histopathology findings of both types of toxaemias coupled to the infrequency of CPI toxaemia might lead to symptomatic misidentification with Type C resulting in therapeutic failure due to habitual administration of CPB anti-toxin which is ineffective against CPI. Therefore in the present study, to generate a composite anti-toxin capable of neutralizing both toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-toxic C terminal binding regions of CPB and CPI, via a flexible glycine linker (G4S) by overlap-extension PCR. The fusion protein was characterized for its therapeutic abilities toward CPI and CPB toxin neutralizations. The r-Cpib was found to be non-toxic and could competitively inhibit binding of CPB to host cell receptors thereby reducing its cytotoxicity. Immunization of mice with r-Cpib generated specific antibodies capable of neutralizing the above toxaemias both in vitro and in vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB, displayed significantly superior protection against the respective toxins while passive challenge of mice with a similar mixture resulted in 83 and 91% protection against CPI and CPB respectively. Alternatively, mice exposed to a mixture of sham sera and native toxins died within 2-3 days. This work thus demonstrates r-Cpib as a novel bivalent fusion protein capable of efficient immunotherapy against C. perfringens CPI and CPB toxaemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

A deimmunized bispecific ligand directed toxin that shows an impressive anti-pancreatic cancer effect in a systemic nude mouse orthotopic model

PubMed Central

Oh, Seunguk; Todhunter, Deborah A.; Panoskaltsis-Mortari, Angela; Buchsbaum, Donald J.; Toma, Shoko; Vallera, Daniel A.

2011-01-01

Objective The objective was to test a bispecific ligand directed toxin (BLT), with reduced immunogenicity for enhanced efficacy in targeting orthotopic pancreatic cancer in vivo. Method A new BLT was created in which both human EGF and IL-4 cytokines were cloned onto the same single chain molecule with deimmunized pseudomonas exotoxin (dEGF4KDEL). Key amino acids dictating B cell generation of neutralizing anti-toxin antibodies were mutated. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were performed to determine whether anti-toxin antibodies were reduced. A genetically altered luciferase MIA PaCa-2 xenograft model was used to image in real time and determine affects on systemic malignant human cancer. BLTs targeting B cells were used as specificity controls. Results dEGF4KDEL was significantly effective following systemic injection against established orthotopic MIA PaCa-2 pancreatic cancer and selectively prevented metastasis. Mutagenesis significantly reduced anti-toxin levels in vivo with no apparent activity loss in vitro. The drug was effective against three human pancreatic cancer lines in vitro, MIA PaCa-2, SW1990, and S2VP10. Conclusions Despite the metastatic nature of the MIA PaCa-2 orthotopic tumor xenografted in nude mice, high percentages of tumors responded to extended dEGFKDEL treatment resulting in significant anti-cancer effects and disease-free survivors. PMID:22258068

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

PubMed

Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

2016-09-11

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

Lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated.

PubMed

Dumas, Eric K; Garman, Lori; Cuthbertson, Hannah; Charlton, Sue; Hallis, Bassam; Engler, Renata J M; Choudhari, Shyamal; Picking, William D; James, Judith A; Farris, A Darise

2017-06-08

A major difference between two currently licensed anthrax vaccines is presence (United Kingdom Anthrax Vaccine Precipitated, AVP) or absence (United States Anthrax Vaccine Adsorbed, AVA) of quantifiable amounts of the Lethal Toxin (LT) component Lethal Factor (LF). The primary immunogen in both vaccine formulations is Protective Antigen (PA), and LT-neutralizing antibodies directed to PA are an accepted correlate of vaccine efficacy; however, vaccination studies in animal models have demonstrated that LF antibodies can be protective. In this report we compared humoral immune responses in cohorts of AVP (n=39) and AVA recipients (n=78) matched 1:2 for number of vaccinations and time post-vaccination, and evaluated whether the LF response contributes to LT neutralization in human recipients of AVP. PA response rates (≥95%) and PA IgG concentrations were similar in both groups; however, AVP recipients exhibited higher LT neutralization ED 50 values (AVP: 1464.0±214.7, AVA: 544.9±83.2, p<0.0001) and had higher rates of LF IgG positivity (95%) compared to matched AVA vaccinees (1%). Multiple regression analysis revealed that LF IgG makes an independent and additive contribution to the LT neutralization response in the AVP group. Affinity purified LF antibodies from two independent AVP recipients neutralized LT and bound to LF Domain 1, confirming contribution of LF antibodies to LT neutralization. This study documents the benefit of including an LF component to PA-based anthrax vaccines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

PubMed

Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

2015-05-01

The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli

PubMed Central

Taxt, Arne M.; Diaz, Yuleima; Aasland, Rein; Clements, John D.; Nataro, James P.; Sommerfelt, Halvor

2016-01-01

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity. PMID:26883587

An Acoustic Plate Mode Sensor for Biowarfare Toxins, Phase II

DTIC Science & Technology

1997-10-01

Biological agents -- such as bacteria , bacterial toxins and viruses -- must be detected rapidly to allow their neutralization or the quick treatment of...Results were comparable. 16 * r Cyclic voltammetry (CV) studies indicate that the monolayers made with the thiodialkyne, D1, which have been photolyzed...Microprocessor system development is ahead of schedule. Preliminary biosensor data is offered using Y. Pestis. The data indi- cates marginal detection

Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects

PubMed Central

Gal, Yoav; Mazor, Ohad; Falach, Reut; Sapoznikov, Anita; Kronman, Chanoch; Sabo, Tamar

2017-01-01

Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor beans), is one of the most lethal toxins known, particularly if inhaled. Ricin is considered a potential biological threat agent due to its high availability and ease of production. The clinical manifestation of pulmonary ricin intoxication in animal models is closely related to acute respiratory distress syndrome (ARDS), which involves pulmonary proinflammatory cytokine upregulation, massive neutrophil infiltration and severe edema. Currently, the only post-exposure measure that is effective against pulmonary ricinosis at clinically relevant time-points following intoxication in pre-clinical studies is passive immunization with anti-ricin neutralizing antibodies. The efficacy of this antitoxin treatment depends on antibody affinity and the time of treatment initiation within a limited therapeutic time window. Small-molecule compounds that interfere directly with the toxin or inhibit its intracellular trafficking may also be beneficial against ricinosis. Another approach relies on the co-administration of antitoxin antibodies with immunomodulatory drugs, thereby neutralizing the toxin while attenuating lung injury. Immunomodulators and other pharmacological-based treatment options should be tailored according to the particular pathogenesis pathways of pulmonary ricinosis. This review focuses on the current treatment options for pulmonary ricin intoxication using anti-ricin antibodies, disease-modifying countermeasures, anti-ricin small molecules and their various combinations. PMID:28972558

Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects.

PubMed

Gal, Yoav; Mazor, Ohad; Falach, Reut; Sapoznikov, Anita; Kronman, Chanoch; Sabo, Tamar

2017-10-03

Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor beans), is one of the most lethal toxins known, particularly if inhaled. Ricin is considered a potential biological threat agent due to its high availability and ease of production. The clinical manifestation of pulmonary ricin intoxication in animal models is closely related to acute respiratory distress syndrome (ARDS), which involves pulmonary proinflammatory cytokine upregulation, massive neutrophil infiltration and severe edema. Currently, the only post-exposure measure that is effective against pulmonary ricinosis at clinically relevant time-points following intoxication in pre-clinical studies is passive immunization with anti-ricin neutralizing antibodies. The efficacy of this antitoxin treatment depends on antibody affinity and the time of treatment initiation within a limited therapeutic time window. Small-molecule compounds that interfere directly with the toxin or inhibit its intracellular trafficking may also be beneficial against ricinosis. Another approach relies on the co-administration of antitoxin antibodies with immunomodulatory drugs, thereby neutralizing the toxin while attenuating lung injury. Immunomodulators and other pharmacological-based treatment options should be tailored according to the particular pathogenesis pathways of pulmonary ricinosis. This review focuses on the current treatment options for pulmonary ricin intoxication using anti-ricin antibodies, disease-modifying countermeasures, anti-ricin small molecules and their various combinations.

Transcutaneous immunization with cross-reacting material CRM(197) of diphtheria toxin boosts functional antibody levels in mice primed parenterally with adsorbed diphtheria toxoid vaccine.

PubMed

Stickings, Paul; Peyre, Marisa; Coombes, Laura; Muller, Sylviane; Rappuoli, Rino; Del Giudice, Giuseppe; Partidos, Charalambos D; Sesardic, Dorothea

2008-04-01

Transcutaneous immunization (TCI) capitalizes on the accessibility and immunocompetence of the skin, elicits protective immunity, simplifies vaccine delivery, and may be particularly advantageous when frequent boosting is required. In this study we examined the potential of TCI to boost preexisting immune responses to diphtheria in mice. The cross-reacting material (CRM(197)) of diphtheria toxin was used as the boosting antigen and was administered alone or together with either one of two commonly used mucosal adjuvants, cholera toxin (CT) and a partially detoxified mutant of heat-labile enterotoxin of Escherichia coli (LTR72). We report that TCI with CRM(197) significantly boosted preexisting immune responses elicited after parenteral priming with aluminum hydroxide-adsorbed diphtheria toxoid (DTxd) vaccine. In the presence of LTR72 as an adjuvant, toxin-neutralizing antibody titers were significantly higher than those elicited by CRM(197) alone and were comparable to the functional antibody levels induced after parenteral booster immunization with the adsorbed DTxd vaccine. Time course study showed that high levels of toxin-neutralizing antibodies persisted for at least 14 weeks after the transcutaneous boost. In addition, TCI resulted in a vigorous antigen-specific proliferative response in all groups of mice boosted with the CRM(197) protein. These findings highlight the promising prospect of using booster administrations of CRM(197) via the transcutaneous route to establish good herd immunity against diphtheria.

Neutralizing Monoclonal Antibodies against Ricin’s Enzymatic Subunit Interfere with Protein Disulfide Isomerase-Mediated Reduction of Ricin Holotoxin In Vitro

PubMed Central

O’Hara, Joanne M.; Mantis, Nicholas J.

2013-01-01

The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin’s enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin’s disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER. PMID:23774033

Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

PubMed

Thullier, Philippe; Avril, Arnaud; Mathieu, Jacques; Behrens, Christian K; Pellequer, Jean-Luc; Pelat, Thibaut

2013-01-01

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection.

PubMed

Roy, Chad J; Brey, Robert N; Mantis, Nicholas J; Mapes, Kelly; Pop, Iliodora V; Pop, Laurentiu M; Ruback, Stephen; Killeen, Stephanie Z; Doyle-Meyers, Lara; Vinet-Oliphant, Heather S; Didier, Peter J; Vitetta, Ellen S

2015-03-24

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.

Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893

PubMed Central

Yu, Li; Mok, Hoyin; Tkaczyk, Christine; Sellman, Bret R.; Wu, Yuling; Oganesyan, Vaheh; Slidel, Tim; Jafri, Hasan; McCarthy, Michael; Bradford, Patricia; Esser, Mark T.

2016-01-01

Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development. PMID:27324766

Targeting Staphylococcus aureus Toxins: A Potential form of Anti-Virulence Therapy

PubMed Central

Kong, Cin; Neoh, Hui-min; Nathan, Sheila

2016-01-01

Staphylococcus aureus is an opportunistic pathogen and the leading cause of a wide range of severe clinical infections. The range of diseases reflects the diversity of virulence factors produced by this pathogen. To establish an infection in the host, S. aureus expresses an inclusive set of virulence factors such as toxins, enzymes, adhesins, and other surface proteins that allow the pathogen to survive under extreme conditions and are essential for the bacteria’s ability to spread through tissues. Expression and secretion of this array of toxins and enzymes are tightly controlled by a number of regulatory systems. S. aureus is also notorious for its ability to resist the arsenal of currently available antibiotics and dissemination of various multidrug-resistant S. aureus clones limits therapeutic options for a S. aureus infection. Recently, the development of anti-virulence therapeutics that neutralize S. aureus toxins or block the pathways that regulate toxin production has shown potential in thwarting the bacteria’s acquisition of antibiotic resistance. In this review, we provide insights into the regulation of S. aureus toxin production and potential anti-virulence strategies that target S. aureus toxins. PMID:26999200

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

PubMed

Nandre, Rahul; Ruan, Xiaosai; Lu, Ti; Duan, Qiangde; Sack, David; Zhang, Weiping

2018-03-01

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTa A14Q -dmLT or CFA/I/II/IV-2xSTa N12S -dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STa N12S , and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa + or LT + ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa + ETEC and LT + ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines. Copyright © 2018 American Society for Microbiology.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

PubMed

Arimitsu, Hideyuki; Sasaki, Keiko; Kohda, Tomoko; Shimizu, Toshiyasu; Tsuji, Takao

2014-11-01

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Comparative performance of a licensed anthrax vaccine versus electroporation based delivery of a PA encoding DNA vaccine in rhesus macaques.

PubMed

Livingston, Brian D; Little, Stephen F; Luxembourg, Alain; Ellefsen, Barry; Hannaman, Drew

2010-01-22

DNA vaccination is a promising immunization strategy that could be applied in the development of vaccines for a variety of prophylactic and therapeutic indications. Utilizing anthrax protective antigen as a model antigen, we demonstrate that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection. Two administrations of a DNA vaccine with electroporation elicited anthrax toxin neutralizing antibody responses in 100% of rhesus macaques. Toxin neutralizing antibodies were sustained for the nearly 1-year study duration and were correlated with protection against subsequent lethal Bacillus anthracis spore challenge. Collectively, electroporation mediated DNA vaccination conferred protection comparable to that observed following vaccination with an FDA approved anthrax vaccine.

Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin.

PubMed

Trott, D L; Yang, M; Gonzalez, J; Larson, A E; Tepp, W H; Johnson, E A; Cook, M E

2009-05-01

The objective of this research project was to determine the usefulness of an egg antibody platform for producing materials for the detection and neutralization of botulinum type A neurotoxin. Yield estimates for detection and neutralizing antibodies produced using methods described were calculated. Antibody specific to botulinum toxoid A (aToxoid) and toxin A (aBoNT/A) was produced by immunizing hens with botulinum toxoid A (toxoid) followed by increasing amounts of botulinum neurotoxin A (BoNT/A) in Freund incomplete adjuvant. Egg yolks were extracted with polyethylene glycol (PEG) for antibody detection and neutralization experiments. A model aToxoid/toxoid immunoassay using only egg yolk antibody was developed and had a detection limit of 1 pg/ml of toxoid. In an indirect enzyme-linked immunosorbent assay of BoNT/A-specific antibody, the aBoNT/A contained more BoNT/A-specific antibody than did the aToxoid, and aBoNT/A was as effective as commercial rabbit antibody. The aToxoid provided no protection against BoNT/A in a standard mouse neutralization assay; however, 1 mg of PEG-extracted aBoNT/A neutralized 4,000 lethal doses of BoNT/A injected intraperitoneally. Based on these results, we calculated that in 1 month one hen could produce more than 100 liters of antibody detection reagents or enough antibody to neutralize approximately 11.6 million mouse lethal doses of botulinum toxin. Utilization of an egg antibody platform is potentially rapid (28 to 70 days) and scalable to kilogram quantities using current egg production facilities with as few as 1,000 hens.

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin’s binding subunit

PubMed Central

Rong, Yinghui; Van Slyke, Greta; Vance, David J.; Westfall, Jennifer; Ehrbar, Dylan

2017-01-01

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α. PMID:28700745

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti-Botulinum Antibody Preparations.

PubMed

Torgeman, Amram; Ozeri, Eyal; Ben David, Alon; Diamant, Eran; Rosen, Osnat; Schwartz, Arieh; Barnea, Ada; Makovitzki, Arik; Mimran, Avishai; Zichel, Ran

2017-05-29

The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper-immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper-sensitivity reactions. New-generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti-botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400-mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab')2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper-immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti-botulinum toxin polyclonal horse F(ab')2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal-based to human-based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

Combination of Two Candidate Subunit Vaccine Antigens Elicits Protective Immunity to Ricin and Anthrax Toxin in Mice

PubMed Central

Vance, David J.; Rong, Yinghui; Brey, Robert N.; Mantis, Nicholas J.

2014-01-01

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population. PMID:25475957

A tripartite fusion, FaeG-FedF-LT(192)A2:B, of enterotoxigenic Escherichia coli (ETEC) elicits antibodies that neutralize cholera toxin, inhibit adherence of K88 (F4) and F18 fimbriae, and protect pigs against K88ac/heat-labile toxin infection.

PubMed

Ruan, Xiaosai; Liu, Mei; Casey, Thomas A; Zhang, Weiping

2011-10-01

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.

Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro.

PubMed

O'Hara, Joanne M; Mantis, Nicholas J

2013-09-30

The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin's enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin's disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER. Copyright © 2013 Elsevier B.V. All rights reserved.

Tetanus

MedlinePlus

... Clostridium tetani that usually live in soil. The bacteria produce a toxin (a chemical or poison that harms ... care unit (ICU). They receive large doses of antibiotics to kill the tetanus bacteria and tetanus antitoxin (a medicine that neutralizes the ...

Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum.

PubMed

Hsu, Po-Yuan; Lee, Kuo-Kau; Hu, Chih-Chuang; Liu, Ping-Chung

2014-09-01

Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies of guinea pig immunoglobulin isotype, idiotype and antiidiotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tirrell, S.M.

1988-01-01

Immunization of Guinea pigs with diphtheria toxoid generated antibodies of the IgG class that were capable of neutralizing native toxin in vivo. Sera from these animals were used to affinity purify idiotypic antibodies (AB1). AB1 vaccines derived from the IgG1 class and from F(ab{prime}){sub 2} of IgG1 + IgG2 (IgG1/2) classes were effective in inducing a syngeneic anti-idiotype (AB2) response. Animals immunized with AB1 consisting of both IgG1/2 did not elicit a detectable AB2 response. Binding of homologous {sup 125}I-F(ab{prime}){sub 2} (AB1) to the antiidiotype was inhibited 90% in the presence of DT.F(ab{prime}){sub 2} derived from preimmune serum or hadmore » no inhibitory effects on the idiotype-antiidiotype interactions. Two groups of outbred guinea pigs were vaccinated with alum absorbed F(ab{prime}){sub 2} of anti-idiotype IgG1/2 (AB2). Of the ten animals inoculated with AB2, three tested positive by RIA against {sup 125}I-DT. Two of the RIA positive sera contained antibodies that neutralized diphtheria toxin in a rabbit intracutaneous assay. Purification of guinea pig IgG by protein A-Sepharose affinity chromatography resulted in the separation of three distinct IgG populations.« less

Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.

PubMed

Thomas, Justin C; O'Hara, Joanne M; Hu, Lei; Gao, Fei P; Joshi, Sangeeta B; Volkin, David B; Brey, Robert N; Fang, Jianwen; Karanicolas, John; Mantis, Nicholas J; Middaugh, C Russell

2013-04-01

There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

PubMed

Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

2015-11-27

Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

PubMed Central

Cheng, Luisa W.; Henderson, Thomas D.; Lam, Tina I.; Stanker, Larry H.

2015-01-01

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-06-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed Central

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-01-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

Mechanisms for Differential Protein Production in Toxin–Antitoxin Systems

PubMed Central

Deter, Heather S.; Jensen, Roderick V.; Mather, William H.; Butzin, Nicholas C.

2017-01-01

Toxin–antitoxin (TA) systems are key regulators of bacterial persistence, a multidrug-tolerant state found in bacterial species that is a major contributing factor to the growing human health crisis of antibiotic resistance. Type II TA systems consist of two proteins, a toxin and an antitoxin; the toxin is neutralized when they form a complex. The ratio of antitoxin to toxin is significantly greater than 1.0 in the susceptible population (non-persister state), but this ratio is expected to become smaller during persistence. Analysis of multiple datasets (RNA-seq, ribosome profiling) and results from translation initiation rate calculators reveal multiple mechanisms that ensure a high antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms include both translational and transcriptional regulation. We classified E. coli type II TA systems into four distinct classes based on the mechanism of differential protein production between toxin and antitoxin. We find that the most common regulation mechanism is translational regulation. This classification scheme further refines our understanding of one of the fundamental mechanisms underlying bacterial persistence, especially regarding maintenance of the antitoxin-to-toxin ratio. PMID:28677629

Use of antibodies specific to defined regions of scorpion. cap alpha. -toxin to study its interaction with its receptor site on the sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ayeb, M.E.; Bahraoui, E.M.; Granier, C.

1986-10-21

Five antibody populations selected by immunoaffinity chromatography for the specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the /sup 125/I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to the antibodies when themore » toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only ..cap alpha..-helix region (residues 23-32) and a ..beta..-turn structure (residues 32-35) are accessible to the respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.« less

Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice.

PubMed

Vance, David J; Rong, Yinghui; Brey, Robert N; Mantis, Nicholas J

2015-01-09

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Effect of Total Cumulative Dose, Number of Treatment Cycles, Interval between Injections, and Length of Treatment on the Frequency of Occurrence of Antibodies to Botulinum Toxin Type A in the Treatment of Muscle Spasticity

ERIC Educational Resources Information Center

Bakheit, Abdel Magid O.; Liptrot, Anthea; Newton, Rachel; Pickett, Andrew M.

2012-01-01

A large cumulative dose of botulinum toxin type A (BoNT-A), frequent injections, a short interval between treatment cycles, and a long duration of treatment have all been suggested, but not confirmed, to be associated with a high incidence of neutralizing antibodies to the neurotoxin. The aim of this study was to investigate whether these…

Repurposing a Prokaryotic Toxin-Antitoxin System for the Selective Killing of Oncogenically Stressed Human Cells.

PubMed

Preston, Mark A; Pimentel, Belén; Bermejo-Rodríguez, Camino; Dionne, Isabelle; Turnbull, Alice; de la Cueva-Méndez, Guillermo

2016-07-15

Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until coexpressed antitoxin partners are degraded in response to stress. Although not evolved to function in eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a synthetic system that becomes active in human cells suffering a specific oncogenic stress. Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin system functions autonomously in human cells, distinguishing those that suffer the oncogenic insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results provide a framework for developing personalized anticancer strategies avoiding off-target effects, a challenge that has been hardly tractable by other means thus far.

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2016-02-01

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein

PubMed Central

Adekar, Sharad P.; Segan, Andrew T.; Chen, Cindy; Bermudez, Rodney; Elias, M. D.; Selling, Bernard H.; Kapadnis, B. P.; Simpson, Lance L.; Simon, Paul M.; Dessain, Scott K.

2011-01-01

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo. PMID:21399689

A toxin-antitoxin module as a target for antimicrobial development.

PubMed

Lioy, Virginia S; Rey, Oscar; Balsa, Dolors; Pellicer, Teresa; Alonso, Juan C

2010-01-01

The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin-antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded TA loci, the labile epsilon antitoxin binds to free zeta toxin and neutralizes it. When the zeta toxin is freed from the epsilon antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged zeta toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the epsilon.zeta interaction can be considered as an attractive antimicrobial agent. Gene epsilon was fused to luc (Luc-epsilon antitoxin) and zeta to the gfp gene (zeta-GFP). Luc-epsilon or epsilon antitoxin neutralizes the toxic effect of the zeta or zeta-GFP toxin. In the absence of the antitoxin, free zeta or zeta-GFP triggers a reversible loss of cell proliferation, but the zetaK46A-GFP variant fails to block growth. Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). To develop the proper controls, molecular dynamics studies were used to predict that the Asp18 and/or Glu22 residues might be relevant for epsilon.zeta interaction. Luc-epsilon efficiently transfers the excited energy to the fluorescent acceptor molecule (zeta-GFP or zetaK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from zeta (D18A) affects Luc-epsilon.zetaD18A K46A-GFP interaction. In this study, we validate the hypothesis that it is possible to disrupt a TA module and offer a novel and unexploited targets to fight against antibiotic-resistant strains. Copyright 2009 Elsevier Inc. All rights reserved.

Gender differences in onabotulinum toxin A dosing for adductor spasmodic dysphonia.

PubMed

Lerner, Michael Z; Lerner, Benjamin A; Patel, Amit A; Blitzer, Andrew

2017-05-01

The objective of this study was to determine the influence of gender on onabotulinum toxin A dosing for the treatment of adductor spasmodic dysphonia symptoms. Retrospective review. A chart review of the senior author's database of botulinum toxin injections was performed. Patients diagnosed with adductor spasmodic dysphonia who received onabotulinum toxin A (BoNTA) injections to the thyroarytenoid muscle for at least 5 years were included for study. Patients who received alternate formulations of botulinum toxin (Myobloc, Dysport, or Xeomin) and patients with alternate diagnoses, such as abductor spasmodic dysphonia, tremor, and oromandibular dystonia, were excluded. The average BoNTA dose was calculated for each patient and statistical analysis was performed comparing the male and female groups. A total of 201 patients (52 males and 149 females) met inclusion criteria. The average follow-up times for the male and female groups were 10.2 ± 3.6 and 11.1 ± 4 years, respectively. The average BoNTA doses for the male and female groups were 0.6 ± 0.42 U and 1.3 ± 1.1 U, respectively. Statistical analysis was performed using an independent samples two-tailed t test yielding a P value of .0000000002. A large effect size was noted with Cohen's d = 0.85. The data from this retrospective chart review reveal a statistically and clinically significant correlation between female gender and higher average BoNTA dose for symptom control in adductor spasmodic dysphonia. Explanations for this observation are speculative and include a possible inverse relationship between optimal BoNTA dose and vocal fold mass and possibly greater neutralizing antibody formation among female patients. 4. Laryngoscope, 127:1131-1134, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems

PubMed Central

Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

2017-01-01

ABSTRACT The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3–4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors. PMID:28060574

Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody.

PubMed

Kang, Changkeun; Han, Dae-Yong; Park, Kwang-Il; Pyo, Min-Jung; Heo, Yunwi; Lee, Hyunkyoung; Kim, Gon Sup; Kim, Euikyung

2014-08-01

Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

PubMed

Orth, Peter; Xiao, Li; Hernandez, Lorraine D; Reichert, Paul; Sheth, Payal R; Beaumont, Maribel; Yang, Xiaoyu; Murgolo, Nicholas; Ermakov, Grigori; DiNunzio, Edward; Racine, Fred; Karczewski, Jerzy; Secore, Susan; Ingram, Richard N; Mayhood, Todd; Strickland, Corey; Therien, Alex G

2014-06-27

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A bioanalytical platform for simultaneous detection and quantification of biological toxins.

PubMed

Weingart, Oliver G; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin's identity and concentration. The system's performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5-1 ng · mL(-1) in buffer or in raw milk.

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

PubMed Central

Torres, J F; Lyerly, D M; Hill, J E; Monath, T P

1995-01-01

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization. PMID:7591115

Anthrax Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

PubMed Central

Dumas, Eric K.; Gross, Timothy; Larabee, Jason; Pate, Lance; Cuthbertson, Hannah; Charlton, Sue; Hallis, Bassam; Engler, Renata J. M.; Collins, Limone C.; Spooner, Christina E.; Chen, Hua; Ballard, Jimmy; James, Judith A.

2017-01-01

ABSTRACT Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP (n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines. PMID:28877928

Sub-Domains of Ricin’s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

PubMed Central

Yermakova, Anastasiya; Vance, David J.; Mantis, Nicholas J.

2012-01-01

The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin. PMID:22984492

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

PubMed

Secore, Susan; Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S; Bodmer, Jean-Luc; Heinrichs, Jon H

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains

PubMed Central

Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R.; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S.; Bodmer, Jean-Luc; Heinrichs, Jon H.

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile. PMID:28125650

Impact of Anti-Shiga Toxin Type 2 (Stx2) Neutralizing Antibody on Colonization and Pathogenesis of Escherichia Coli O157:H7 in Mice

DTIC Science & Technology

2010-03-17

Infect Immun 56:3099-3109. 86. Fontaine, R. E., S. Arnon, W. T. Martin , T. M. Vernon, Jr., E. J. Gangarosa, J. J. Farmer, 3rd, A. B. Moran, J. H...Bernalier-Donadille, M. Vareille, A. P. Gobert, and C. Martin . 2009. Human microbiota-secreted factors inhibit shiga toxin 303 synthesis by...infective defences in the gastrointestinal tract. Best Pract Res Clin Gastroenterol 17:755-773. 33. Griffin, P. M., L. C. Olmstead, and R. E. Petras

Scorpion-Toxin Mimics of CD4 in Complex with Human Immunodeficiency Virus gp120: Crystal Structures, Molecular Mimicry, and Neutralization Breadth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Chih-chin; Stricher, Francois; Martin, Loic

The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increasedmore » neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.« less

OBSERVATIONS ON THE PRESENCE OF A TOXIC SUBSTANCE IN THE BLOOD AND URINE OF PATIENTS WITH SCARLET FEVER

PubMed Central

Trask, James D.; Blake, Francis G.

1924-01-01

A series of observations on the blood of patients acutely ill with scarlet fever has shown that a toxic substance can be demonstrated in the serum by means of intracutaneous injections of the serum in persons who have not had scarlet fever and whose serums fail to blanch the rash in scarlet fever. The reaction caused by this substance consists of a bright red local erythema, varying from 20 to 70 mm. in diameter, of 1 to 4 days duration. The severer reactions are moderately indurated and tender, and are followed bypigmentation and desquamation. Control injections in persons whose serums blanch the rash in scarlet fever cause no reaction. The toxic substance is not neutralized by mixture with a human serum which gives a negative blanching test but is readily neutralized by a human serum which gives a positive blanching test. It is not neutralized by normal horse serum, but is completely neutralized by Dochez's scarlatinal antistreptococcic serum. In a limited number of observations on the urine of patients with scarlet fever a similar toxic substance has been found in two out of five cases studied. Since the toxic substance described appears to resemble the toxic substance found in the filtrates of scarlatinal hemolytic streptococcus cultures by Dick and Dick and since it is neutralized not only by a blanching human serum but also by Dochez's scarlatinal antistreptococcic horse serum, the experiments reported support the conception that scarlet fever is a local infection of the throat by a particular type of Streptococcus hæmolyticus capable of producing a toxin which is absorbed and is the cause of the general manifestations of the disease. PMID:19868926

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

PubMed Central

Ferreira, Marcos Roberto A.; Moreira, Gustavo Marçal S. G.; da Cunha, Carlos Eduardo P.; Mendonça, Marcelo; Salvarani, Felipe M.; Moreira, Ângela N.; Conceição, Fabricio R.

2016-01-01

Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals. PMID:27879630

A Novel Biomimetic Nanosponge Protects the Retina from the Enterococcus faecalis Cytolysin.

PubMed

LaGrow, Austin L; Coburn, Phillip S; Miller, Frederick C; Land, Craig; Parkunan, Salai Madhumathi; Luk, Brian T; Gao, Weiwei; Zhang, Liangfang; Callegan, Michelle C

2017-01-01

Intraocular infections are a potentially blinding complication of common ocular surgeries and traumatic eye injuries. Bacterial toxins synthesized in the eye can damage intraocular tissue, often resulting in poor visual outcomes. Enteroccocus faecalis causes blinding infections and is responsible for 8 to 17% of postoperative endophthalmitis cases. These infections are increasingly difficult to treat due to the emergence of multidrug-resistant strains. Virulent E. faecalis isolates secrete a pore-forming bicomponent cytolysin that contributes to retinal tissue damage during endophthalmitis. We hypothesized that a biomimetic nanosponge, which mimics erythrocytes, might adsorb subunits of the cytolysin and reduce retinal damage, protecting vision. To test the efficacy of nanosponges in neutralizing the cytolysin in vitro , hemoglobin release assays were performed on culture supernatants from cytolysin-producing E. faecalis with and without preincubation with nanosponges. Treatment with nanosponges for 30 min reduced hemolytic activity by ~70%. To determine whether nanosponges could neutralize the cytolysin in vivo , electroretinography was performed on mice 24 h after intravitreal injection with cytolysin-containing supernatants treated with nanosponges. Pretreatment of cytolysin-containing supernatants with nanosponges increased the A-wave retention from 12.2% to 65.5% and increased the B-wave retention from 21.0% to 77.0%. Histology revealed that in nanosponge-treated eyes, retinas remained intact and attached, with little to no damage. Rabbit nanosponges were also nontoxic and noninflammatory when injected into mouse eyes. In an experimental murine model of E. faecalis endophthalmitis, injection of nanosponges into the vitreous 6 h after infection with a wild-type cytolysin-producing strain increased A-wave retention from 5.9% to 31% and increased B-wave retention from 12.6% to 27.8%. Together, these results demonstrated that biomimetic nanosponges neutralized cytolysin activity and protected the retinas from damage. These results suggest that this novel strategy might also protect eyes from the activities of pore-forming toxins of other virulent ocular bacterial pathogens. IMPORTANCE Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E. faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E. faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections.

Recent advances in the development of vaccines against ricin.

PubMed

Brey, Robert N; Mantis, Nicholas J; Pincus, Seth H; Vitetta, Ellen S; Smith, Leonard A; Roy, Chad J

2016-05-03

Several promising subunit vaccines against ricin toxin (RT) have been developed during the last decade and are now being tested for safety and immunogenicity in humans and for efficacy in nonhuman primates. The incentive to develop a preventive vaccine as a countermeasure against RT use as a bioweapon is based on the high toxicity of RT after aerosol exposure, its environmental stability, abundance, and ease of purification. RT is the second most lethal biological toxin and is considered a "universal toxin" because it can kill all eukaryotic cells through binding to ubiquitous cell surface galactosyl residues. RT has two subunits conjoined by a single disulfide linkage: RTB, which binds galactosyl residues and RTA which enzymatically inactivates ribosomes intracellularly by cleavage ribosomal RNA. Attenuation of toxicity by elimination of the active site or introduction of other structural mutations of RTA has generated two similar clinical subunit vaccine candidates which induce antibodies in both humans and nonhuman primates. In rhesus macaques, inhaled RT causes rapid lung necrosis and fibrosis followed by death. After parenteral vaccination with RTA vaccine, macaques can be protected against aerosol RT exposure, suggesting that circulating antibodies can protect lung mucosa. Vaccination induces RT-neutralizing antibodies, the most likely correlate of protection. Macaques responded to conformational determinants in an RTA vaccine formulation, indicating preservation of RTA structure during initial manufacture. Comparative mapping studies have also demonstrated that macaques and humans recognize the same epitopes, significant in the study of macaques as a model during development of vaccines which cannot be tested for efficacy in humans.

Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis

PubMed Central

de la Cueva-Méndez, Guillermo; Mills, Anthony D.; Clay-Farrace, Lorena; Díaz-Orejas, Ramón; Laskey, Ronald A.

2003-01-01

Plasmid R1 inhibits growth of bacteria by synthesizing an inhibitor of cell proliferation, Kid, and a neutralizing antidote, Kis, which binds tightly to the toxin. Here we report that this toxin and antidote, which have evolved to function in bacteria, also function efficiently in a wide range of eukaryotes. Kid inhibits cell proliferation in yeast, Xenopus laevis and human cells, whilst Kis protects. Moreover, we show that Kid triggers apoptosis in human cells. These effects can be regulated in vivo by modulating the relative amounts of antidote and toxin using inducible eukaryotic promoters for independent transcriptional control of their genes. These findings allow highly regulatable, selective killing of eukaryotic cells, and could be applied to eliminate cancer cells or specific cell lineages in development. PMID:12514130

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

PubMed Central

Wagner, Leslie; Verma, Anita; Meade, Bruce D.; Reiter, Karine; Narum, David L.; Brady, Rebecca A.; Little, Stephen F.

2012-01-01

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies. PMID:22815152

Snake-venom resistance as a mammalian trophic adaptation: lessons from didelphid marsupials.

PubMed

Voss, Robert S; Jansa, Sharon A

2012-11-01

Mammals that prey on venomous snakes include several opossums (Didelphidae), at least two hedgehogs (Erinaceidae), several mongooses (Herpestidae), several mustelids, and some skunks (Mephitidae). As a group, these taxa do not share any distinctive morphological traits. Instead, mammalian adaptations for ophiophagy seem to consist only in the ability to resist the toxic effects of snake venom. Molecular mechanisms of venom resistance (as indicated by biochemical research on opossums, mongooses, and hedgehogs) include toxin-neutralizing serum factors and adaptive changes in venom-targeted molecules. Of these, toxin-neutralizing serum factors have received the most research attention to date. All of the toxin-neutralizing serum proteins discovered so far in both opossums and mongooses are human α1B-glycoprotein homologs that inhibit either snake-venom metalloproteinases or phospholipase A(2) myotoxins. By contrast, adaptive changes in venom-targeted molecules have received far less attention. The best-documented examples include amino-acid substitutions in mongoose nicotinic acetylcholine receptor that inhibit binding by α-neurotoxins, and amino-acid substitutions in opossum von Willebrand factor (vWF) that are hypothesized to weaken the bond between vWF and coagulopathic C-type lectins. Although multiple mechanisms of venom resistance are known from some species, the proteomic complexity of most snake venoms suggests that the evolved biochemical defences of ophiophagous mammals are likely to be far more numerous than currently recognized. Whereas most previous research in this field has been motivated by the potential for medical applications, venom resistance in ophiophagous mammals is a complex adaptation that merits attention from comparative biologists. Unfortunately, evolutionary inference is currently limited by ignorance about many relevant facts that can only be provided by future research. © 2012 The Authors. Biological Reviews © 2012 Cambridge Philosophical Society.

Structure-function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN-MNT toxin-antitoxin system.

PubMed

Jia, Xuanyan; Yao, Jianyun; Gao, Zengqiang; Liu, Guangfeng; Dong, Yu-Hui; Wang, Xiaoxue; Zhang, Heng

2018-05-04

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite R X 4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the R X 4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family. © 2018 Jia et al.

Multiple Diphtheria Antigen-Antibody Systems Investigated by Passive Haemagglutination Techniques and Other Methods

PubMed Central

Fulthorpe, A. J.

1962-01-01

A fair degree of correlation has been found between the in vivo antitoxin content of sera from horses immunized with crude Corynebacterium diphtheriae culture filtrates and the direct agglutinin titre of the sera when tested with sheep cells sensitized with diphtheria toxoid. Haemagglutination inhibition tests at the LA level of test with the same sera showed some rather large discrepancies from the in vivo and further tests with special agglutinin inhibiting toxins suggested that specific antitoxin free from other accessory antibodies might be non-agglutinating, and therefore not titratable by haemagglutination inhibition. The phosphate-stable, pepsin-stable and trypsin-stable antigens isolated from culture filtrates of C. diphtheriae were found to contain extremely small quantities of specific toxoid, and cross titration of each of the three antigen preparations showed that there was very little contamination by other antigens within the group. Absorption of diphtheria antiserum with red cells sensitized with each of the three accessory antigens individually, showed that the antibodies were highly specific and distinct. Absorption of diphtheria antiserum with a mixture of red cells sensitized with the three different antigens removed all demonstrable accessory antibodies, and the absorbed serum would no longer agglutinate cells sensitized with complete diphtheria toxoid. The absorbed serum, however, retained a large proportion of its neutralizing capacity for diphtheria toxin, when titrated in vivo. Titration of each of the accessory antibodies in a number of horse sera by haemagglutination inhibition demonstrated a correlation between the values for the accessory antibodies to the phosphate-stable and pepsin-stable antigens, but no correlation with the values for the antibody to the trypsin-stable antigen, when compared with results of the flocculation test. The relative proportions of diphtheria toxin and of the phosphate-stable and pepsin-stable antigens remained constant at all stages of purification of a filtrate including several recrystallization procedures. However, the concentration of the trypsin-stable antigen declined steadily during the process. The relative proportions of toxin, phosphate-stable and pepsin-stable antigens in several crude culture filtrates were constant under reasonable conditions of storage and very varied where deterioration had occurred. These three antigens also maintained a constant ratio in growing culture, the trypsin-stable antigen did not. PMID:13895880

The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa.

PubMed

Suzuki, C; Nikkuni, S

1994-01-28

A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl. The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea. Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity. The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta. Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214. The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin. The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria.

PubMed

Lee, Ki-Young; Lee, Bong-Jin

2016-10-22

Bacterial toxin-antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein-protein interactions. Accumulating knowledge about the structure-function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems.

Induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant Clostridium perfringens toxoids.

PubMed

Jiang, Zhigang; De, Yanyan; Chang, Jitao; Wang, Fang; Yu, Li

2014-11-01

Cattle enterotoxemia caused by Clostridium perfringens toxins is a noncontagious, sporadic, and fatal disease characterized by sudden death. Strategies for controlling and preventing cattle enterotoxemia are based on systematic vaccination of herds with toxoids. Because the process of producing conventional clostridial vaccines is dangerous, expensive, and time-consuming, the prospect of recombinant toxoid vaccines against diseases caused by C. perfringens toxins is promising. In this study, nontoxic recombinant toxoids derived from α-, β- and ε-toxins of C. perfringens, namely, rCPA247-370 , rCPB and rEtxHP, respectively, were expressed in Escherichia coli. High levels of specific IgG antibodies and neutralizing antibodies against the toxins were detected in sera from calves vaccinated with either a single recombinant toxoid or a mixed cocktail of all three recombinant toxoids, indicating the potential of these recombinant toxoids to provide calves with protective immunity against enterotoxemia caused by C. perfringens. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

PubMed

Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan

2014-05-19

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability. Copyright © 2014 Elsevier Ltd. All rights reserved.

Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection.

PubMed

Zhang, Yue; Zhang, Jianhua; Chen, Wansong; Angsantikul, Pavimol; Spiekermann, Kevin A; Fang, Ronnie H; Gao, Weiwei; Zhang, Liangfang

2017-10-10

We reported an erythrocyte membrane-coated nanogel (RBC-nanogel) system with combinatorial antivirulence and responsive antibiotic delivery for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. RBC membrane was coated onto the nanogel via a membrane vesicle templated in situ gelation process, whereas the redox-responsiveness was achieved by using a disulfide bond-based crosslinker. We demonstrated that the RBC-nanogels effectively neutralized MRSA-associated toxins in extracellular environment and the toxin neutralization in turn promoted bacterial uptake by macrophages. In intracellular reducing environment, the RBC-nanogels showed an accelerated drug release profile, which resulted in more effective bacterial inhibition. When added to the macrophages infected with intracellular MRSA bacteria, the RBC-nanogels significantly inhibited bacterial growth compared to free antibiotics and non-responsive nanogel counterparts. These results indicate the great potential of the RBC-nanogel system as a new and effective antimicrobial agent against MRSA infection. Copyright © 2017 Elsevier B.V. All rights reserved.

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

PubMed Central

Weingart, Oliver G.; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk. PMID:22438766

Brown spider dermonecrotic toxin directly induces nephrotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaim, Olga Meiri; Sade, Youssef Bacila; Bertoni da Silveira, Rafael

2006-02-15

Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceousmore » material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from culture substratum. In addition, dermonecrotic toxin treatment of MDCK cells changed their viability evaluated by XTT and Neutral-Red Uptake methodologies. The present results point to brown spider dermonecrotic toxin cytotoxicity upon renal structures in vivo and renal cells in vitro and provide experimental evidence that this brown spider toxin is directly involved in nephrotoxicity evoked during Loxosceles spider venom accidents.« less

Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

PubMed Central

Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

2013-01-01

Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (K D values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

Intrinsic and chemically produced microheterogeneity of Staphylococcus aureus enterotoxin type C.

PubMed Central

Metzger, J F; Johnson, A D; Spero, L

1975-01-01

Staphylococcus aureus enterotoxins C1 (SEC1) and C2 (SEC2) produced from 50-liter quantities of crude culture supernatants were purified chromatographically in a neutral or acid milieu. Microheterogenity of SEC1 was markedly increased by treatment of the purified toxin with alkali, and new more acidic charged species appeared. SEC2 was more heterogenous than any of the other S. aureus enterotoxins and was affected only slightly by treatment with alkali. Prolonged incubation of the organism during production of the SEC2 produced changes in charged species that may be related to a bacterial deamidase, since similar changes were not seen with alkaline treatment of the purified toxin. Although SEC1 and SEC2 showed complete identity immunologically, they are separate, distinct toxins, and alkali treatment of SEC1 did not produce SEC2. Images PMID:237837

Use of monoclonal antibodies in the sensitive detection and neutralization of botulinum neurotoxin serotype B

USDA-ARS?s Scientific Manuscript database

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed sensitive electrochemiluminescent (ECL) i...

Immunization of Mice with Anthrax Protective Antigen Limits Cardiotoxicity but Not Hepatotoxicity Following Lethal Toxin Challenge.

PubMed

Devera, T Scott; Prusator, Dawn K; Joshi, Sunil K; Ballard, Jimmy D; Lang, Mark L

2015-06-25

Protective immunity against anthrax is inferred from measurement of vaccine antigen-specific neutralizing antibody titers in serum samples. In animal models, in vivo challenges with toxin and/or spores can also be performed. However, neither of these approaches considers toxin-induced damage to specific organ systems. It is therefore important to determine to what extent anthrax vaccines and existing or candidate adjuvants can provide organ-specific protection against intoxication. We therefore compared the ability of Alum, CpG DNA and the CD1d ligand α-galactosylceramide (αGC) to enhance protective antigen-specific antibody titers, to protect mice against challenge with lethal toxin, and to block cardiotoxicity and hepatotoxicity. By measurement of serum cardiac Troponin I (cTnI), and hepatic alanine aminotransferase (ALT), and aspartate aminotransferase (AST), it was apparent that neither vaccine modality prevented hepatic intoxication, despite high Ab titers and ultimate survival of the subject. In contrast, cardiotoxicity was greatly diminished by prior immunization. This shows that a vaccine that confers survival following toxin exposure may still have an associated morbidity. We propose that organ-specific intoxication should be monitored routinely during research into new vaccine modalities.

Fatal Clostridium botulinum toxicosis in eleven Holstein cattle fed round bale barley haylage.

PubMed

Kelch, W J; Kerr, L A; Pringle, J K; Rohrbach, B W; Whitlock, R H

2000-09-01

Twenty-two lactating Holstein cattle in Tennessee had clinical signs of intoxication with preformed Clostridium botulinum toxin. These signs included weakness, paralysis of the tongue and chest muscles, abdominal breathing, and, in 11 of the 22 cows, death. Differential diagnoses included hypocalcemia, hypomagnesemia, carbohydrate overload, and several toxicoses including mycotoxin, lead, nitrate, organophosphate, atropine or atropine-like alkaloid, and botulism. A diagnosis of botulism by the ingestion of preformed C. botulinum type B toxin was made by eliminating these other diseases, by finding C. botulinum type B spores in 3 bales of round bale barley haylage fed to these cattle, and by isolating preformed type B toxin from 1 of the 3 bales. Confirmation of the toxin type was made by demonstrating mouse lethality by intraperitoneal injection of specimen extracts with neutralization by C. botulinum type B antitoxin. The haylage, harvested green and encased in black plastic bags to facilitate fermentation, was presumably contaminated by the botulinum toxin when fermentation failed to produce enough acid to lower the pH to 4.5, the pH below which C. botulinum growth is inhibited. Farmers and ranchers who use round hay balers to produce haylage should be alert to this potential problem.

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

PubMed

Kashiwagi, T; Kunishima, N; Suzuki, C; Tsuchiya, F; Nikkuni, S; Arata, Y; Morikawa, K

1997-01-15

Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

PubMed

Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

2009-11-01

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

Lipoproteins/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides

PubMed Central

de Tejada, Guillermo Martinez; Heinbockel, Lena; Ferrer-Espada, Raquel; Heine, Holger; Alexander, Christian; Bárcena-Varela, Sergio; Goldmann, Torsten; Correa, Wilmar; Wiesmüller, Karl-Heinz; Gisch, Nicolas; Sánchez-Gómez, Susana; Fukuoka, Satoshi; Schürholz, Tobias; Gutsmann, Thomas; Brandenburg, Klaus

2015-01-01

Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept® which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP. PMID:26390973

Experimentally determined soil organic matter-water sorption coefficients for different classes of natural toxins and comparison with estimated numbers.

PubMed

Schenzel, Judith; Goss, Kai-Uwe; Schwarzenbach, René P; Bucheli, Thomas D; Droge, Steven T J

2012-06-05

Although natural toxins, such as mycotoxins or phytoestrogens are widely studied and were recently identified as micropollutants in the environment, many of their environmentally relevant physicochemical properties have not yet been determined. Here, the sorption affinity to Pahokee peat, a model sorbent for soil organic matter, was investigated for 29 mycotoxins and two phytoestrogens. Sorption coefficients (K(oc)) were determined with a dynamic HPLC-based column method using a fully aqueous mobile phase with 5 mM CaCl(2) at pH 4.5. Sorption coefficients varied from less than 10(0.7) L/kg(oc) (e.g., all type B trichothecenes) to 10(4.0) L/kg(oc) (positively charged ergot alkaloids). For the neutral compounds the experimental sorption data set was compared with predicted sorption coefficients using various models, based on molecular fragment approaches (EPISuite's KOCWIN or SPARC), poly parameter linear free energy relationship (pp-LFER) in combination with predicted descriptors, and quantum-chemical based software (COSMOtherm)). None of the available models was able to adequately predict absolute K(oc) numbers and relative differences in sorption affinity for the whole set of neutral toxins, largely because mycotoxins exhibit highly complex structures. Hence, at present, for such compounds fast and consistent experimental techniques for determining sorption coefficients, as the one used in this study, are required.

Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

PubMed

Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

2011-01-01

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus.

PubMed

Laustsen, Andreas H; Engmark, Mikael; Clouser, Christopher; Timberlake, Sonia; Vigneault, Francois; Gutiérrez, José María; Lomonte, Bruno

2017-01-01

Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A 2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A 2 found in M. nigrocinctus venoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

PubMed

Kerner, Katharina; Bridger, Philip S; Köpf, Gabriele; Fröhlich, Julia; Barth, Stefanie; Willems, Hermann; Bauerfeind, Rolf; Baljer, Georg; Menge, Christian

2015-04-10

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Evaluation of in vivo and in vitro biological activity of a Vibrio cholerae 01 hemolysin.

PubMed

Arellano Galindo, José; Rodriquez Angeles, María Guadalupe; Guadarrama, Norma Valázquez; Esteban, Enrique Santos; Cerezo, Silvia Giono

2007-01-01

To evaluate the hemolysin effect by ileal loop model produced by Vibrio cholerae O1 strains, compared with the cellular lysis or cytotoxic activity (CA) observed in cell culture. We studied nine V. cholerae O1 strains, obtained during the Mexican outbreak of cholera (1990-1993), which had CA in Vero and CHO cells. Hemolysin was monitored with the hemolysis test. Titers of CA were calculated by CD50, and the association between CA and cholera toxin (CT) production was discarded by means of neutralization tests using an anti-CT polyclonal antibody. The CT production was measured with ELISA test. The LAL assay was performed in order to study relationships between the CA and bacterial lipopolysaccharide. Strains with CA were evaluated in rabbit and rat ileal loop models; hemorrhagic fluid was also measured. Tissues from ileal loop were included in paraffin to detect intestinal epithelial damage. The hemolysin CA was not neutralized with the anti-CT polyclonal antibody. However, the associated factor of CA was heat labile. CA in cell cultures was not related to the bacterial lipopolysaccharide. The ileal loop test exhibited the presence of hemorrhagic tissue with inflammation. The V. cholerae O1 strains isolated were able to secrete hemolysin which, in turn, caused CA in cell cultures and produced the hemorrhagic and inflammatory effects observed in the ileal loop of rabbit and rat models.

Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241.

PubMed

Palmer, John; Bell, Matt; Darko, Christian; Barnewall, Roy; Keane-Myers, Andrea

2014-11-01

In the past decade, several Bacillus cereus strains have been isolated from otherwise healthy individuals who succumbed to bacterial pneumonia presenting symptoms resembling inhalational anthrax. One strain was indistinguishable from B. cereus G9241, previously cultured from an individual who survived a similar pneumonia-like illness and which was shown to possess a complete set of plasmid-borne anthrax toxin-encoding homologs. The finding that B. cereus G9241 pathogenesis in mice is dependent on pagA1-derived protective antigen (PA) synthesis suggests that an anthrax toxin-based vaccine may be effective against this toxin-encoding B. cereus strain. Dunkin Hartley guinea pigs were immunized with protein- and DNA-based anthrax toxin-based vaccines, immune responses were evaluated and survival rates were calculated after lethal aerosol exposure with B. cereus G9241 spores. Each vaccine induced seroconversion with the protein immunization regimen eliciting significantly higher serum levels of antigen-specific antibodies at the prechallenge time-point compared with the DNA-protein prime-boost immunization schedule. Complete protection against lethal challenge was observed in all groups with a detectable prechallenge serum titer of toxin neutralizing antibodies. For the first time, we demonstrated that the efficacy of fully defined anthrax toxin-based vaccines was protective against lethal B. cereus G9241 aerosol challenge in the guinea pig animal model. Published 2014. This article is a US Government work and is in the public domain in the USA.

Clostridium difficile shows no trade-off between toxin and spore production within the human host.

PubMed

Blanco, Natalia; Walk, Seth; Malani, Anurag N; Rickard, Alexander; Benn, Michele; Eisenberg, Marisa; Zhang, Min; Foxman, Betsy

2018-05-01

This study aimed to describe the correlation between Clostridium difficile spore and toxin levels within the human host. In addition, we assessed whether overgrowth of Candida albicans modified this association. We measured toxin, spore and Candida albicans levels among 200 successively collected stool samples that tested positive for C. difficile, and PCR ribotyped these C. difficile isolates. Analysis of variance and linear regression were used to test the association between spore and toxin levels. Kruskal-Wallis tests and t-tests were used to compare the association between spore or toxin levels and host, specimen, or pathogen characteristics. C. difficile toxin and spore levels were positively associated (P<0.001); this association did not vary significantly with C. albicans overgrowth [≥5 logs of C. albicans colony-forming units (c.f.u.) g -1 ]. However, ribotypes 027 and 078-126 were significantly associated with higher levels of toxin and spores, and C. albicans overgrowth. The strong positive association observed between in vivo levels of C. difficile toxin and spores suggests that patients with more severe C. difficile infections may have increased spore production, enhancing C. difficile transmission. Although, on average, spore levels were higher in toxin-positive samples than in toxin-negative/PCR-positive samples, spores were found in almost all toxin-negative samples. The ubiquity of spore production among toxin-negative and formed stool samples emphasizes the importance of following infection prevention and control measures for all C. difficile-positive patients during their entire hospital stay.

A Novel Biomimetic Nanosponge Protects the Retina from the Enterococcus faecalis Cytolysin

PubMed Central

LaGrow, Austin L.; Coburn, Phillip S.; Miller, Frederick C.; Land, Craig; Parkunan, Salai Madhumathi; Luk, Brian T.; Gao, Weiwei; Zhang, Liangfang

2017-01-01

ABSTRACT Intraocular infections are a potentially blinding complication of common ocular surgeries and traumatic eye injuries. Bacterial toxins synthesized in the eye can damage intraocular tissue, often resulting in poor visual outcomes. Enteroccocus faecalis causes blinding infections and is responsible for 8 to 17% of postoperative endophthalmitis cases. These infections are increasingly difficult to treat due to the emergence of multidrug-resistant strains. Virulent E. faecalis isolates secrete a pore-forming bicomponent cytolysin that contributes to retinal tissue damage during endophthalmitis. We hypothesized that a biomimetic nanosponge, which mimics erythrocytes, might adsorb subunits of the cytolysin and reduce retinal damage, protecting vision. To test the efficacy of nanosponges in neutralizing the cytolysin in vitro, hemoglobin release assays were performed on culture supernatants from cytolysin-producing E. faecalis with and without preincubation with nanosponges. Treatment with nanosponges for 30 min reduced hemolytic activity by ~70%. To determine whether nanosponges could neutralize the cytolysin in vivo, electroretinography was performed on mice 24 h after intravitreal injection with cytolysin-containing supernatants treated with nanosponges. Pretreatment of cytolysin-containing supernatants with nanosponges increased the A-wave retention from 12.2% to 65.5% and increased the B-wave retention from 21.0% to 77.0%. Histology revealed that in nanosponge-treated eyes, retinas remained intact and attached, with little to no damage. Rabbit nanosponges were also nontoxic and noninflammatory when injected into mouse eyes. In an experimental murine model of E. faecalis endophthalmitis, injection of nanosponges into the vitreous 6 h after infection with a wild-type cytolysin-producing strain increased A-wave retention from 5.9% to 31% and increased B-wave retention from 12.6% to 27.8%. Together, these results demonstrated that biomimetic nanosponges neutralized cytolysin activity and protected the retinas from damage. These results suggest that this novel strategy might also protect eyes from the activities of pore-forming toxins of other virulent ocular bacterial pathogens. IMPORTANCE Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E. faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E. faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections. PMID:29202038

Anthrax biosensor, protective antigen ion channel asymmetric blockade.

PubMed

Halverson, Kelly M; Panchal, Rekha G; Nguyen, Tam L; Gussio, Rick; Little, Stephen F; Misakian, Martin; Bavari, Sina; Kasianowicz, John J

2005-10-07

The significant threat posed by biological agents (e.g. anthrax, tetanus, botulinum, and diphtheria toxins) (Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. M., Russell, P. K., and Tonat, K. (2002) J. Am. Med. Assoc. 287, 2236-2252) requires innovative technologies and approaches to understand the mechanisms of toxin action and to develop better therapies. Anthrax toxins are formed from three proteins secreted by fully virulent Bacillus anthracis, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). Here we present electrophysiological measurements demonstrating that full-length LF and EF convert the current-voltage relationship of the heptameric PA63 ion channel from slightly nonlinear to highly rectifying and diode-like at pH 6.6. This effect provides a novel method for characterizing functional toxin interactions. The method confirms that a previously well characterized PA63 monoclonal antibody, which neutralizes anthrax lethal toxin in animals in vivo and in vitro, prevents the binding of LF to the PA63 pore. The technique can also detect the presence of anthrax lethal toxin complex from plasma of infected animals. The latter two results suggest the potential application of PA63 nanopore-based biosensors in anthrax therapeutics and diagnostics.

Management of tetanus complication

NASA Astrophysics Data System (ADS)

Somia, I. K. A.

2018-03-01

The mortality rate of tetanus is still high; it is because of various complications due to muscle spasms, autonomic dysfunction, as well as due to prolonged critical care. Management of tetanus with its complications is in intensive care facilities. Management goals include stopping toxin production, neutralization of unbound toxin, management of the airway, muscle spasm control, treatment of autonomic dysfunction and general supportive management. Currently, diazepam is still an effective medication to control of muscle spasm and rigidity. Therapy for autonomic dysfunction that supported by evidence is MgSO4. Also, general supportive management for long-term care remains necessary to prevent other complications such as thromboembolism, infection, malnutrition, and others.

Progress and Challenges Associated with the Development of Ricin Toxin Subunit Vaccines

PubMed Central

Vance, David J.; Mantis, Nicholas J.

2016-01-01

Summary The past several years have seen major advances in the development of a safe and efficacious ricin toxin vaccine, including the completion of two Phase I clinical trials with two different recombinant A subunit (RTA)-based vaccines: RiVax™ and RVEc™ adsorbed to aluminum salt adjuvant, as well as a non-human primate study demonstrating that parenteral immunization with RiVax elicits a serum antibody response that was sufficient to protect against a lethal dose aerosolized ricin exposure. One of the major obstacles moving forward is assessing vaccine efficacy in humans, when neither ricin-specific serum IgG endpoint titers nor toxin-neutralizing antibody levels are accepted as definitive predictors of protective immunity. In this review we summarize ongoing efforts to leverage recent advances in our understanding of RTA-antibody interactions at the structural level to develop novel assays to predict vaccine efficacy in humans. PMID:26998662

Progress and challenges associated with the development of ricin toxin subunit vaccines.

PubMed

Vance, David J; Mantis, Nicholas J

2016-09-01

The past several years have seen major advances in the development of a safe and efficacious ricin toxin vaccine, including the completion of two Phase I clinical trials with two different recombinant A subunit (RTA)-based vaccines: RiVax™ and RVEc™ adsorbed to aluminum salt adjuvant, as well as a non-human primate study demonstrating that parenteral immunization with RiVax elicits a serum antibody response that was sufficient to protect against a lethal dose aerosolized ricin exposure. One of the major obstacles moving forward is assessing vaccine efficacy in humans, when neither ricin-specific serum IgG endpoint titers nor toxin-neutralizing antibody levels are accepted as definitive predictors of protective immunity. In this review we summarize ongoing efforts to leverage recent advances in our understanding of RTA-antibody interactions at the structural level to develop novel assays to predict vaccine efficacy in humans.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sberro, Hila; Leavitt, Azita; Kiro, Ruth

Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicitymore » is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.« less

Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations.

PubMed

Ghosh, Anirban; Baltekin, Özden; Wäneskog, Marcus; Elkhalifa, Dina; Hammarlöf, Disa L; Elf, Johan; Koskiniemi, Sanna

2018-05-02

Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts. © 2018 The Authors.

AtaT blocks translation initiation by N-acetylation of the initiator tRNAfMet.

PubMed

Jurėnas, Dukas; Chatterjee, Sneha; Konijnenberg, Albert; Sobott, Frank; Droogmans, Louis; Garcia-Pino, Abel; Van Melderen, Laurence

2017-06-01

Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNA fMet , but no other aminoacyl-tRNAs, including the elongator Met-tRNA Met . We demonstrate that once acetylated, AcMet-tRNA fMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNA fMet as a prime target to efficiently block cell growth.

Ricin-Holotoxin-Based Vaccines: Induction of Potent Ricin-Neutralizing Antibodies.

PubMed

Sabo, Tamar; Kronman, Chanoch; Mazor, Ohad

2016-01-01

Ricin is one of the most potent and lethal toxins known to which there is no available antidote. Currently, the most promising therapy is based on neutralizing antibodies elicited by active vaccination or given passively. Here, detailed protocols are provided for the production of two ricin holotoxin-based vaccines: monomerized subunit-based vaccine, and a formaldehyde-based ricin toxoid vaccine. Both vaccines were found to be stable with no toxic activity reversion even after long-term storage while eliciting high anti-ricin antibody titers possessing a potent neutralizing activity. The use of these vaccines is highly suitable for both the production of sera that can be used in passive protection experiments and immunization aimed to isolate potent anti-ricin monoclonal antibodies.

Harnessing the glucosyltransferase activities of Clostridium difficile for functional studies of toxins A and B.

PubMed

Darkoh, Charles; Kaplan, Heidi B; Dupont, Herbert L

2011-08-01

The incidence of Clostridium difficile infection (CDI) has been increasing within the last decade. Pathogenic strains of C. difficile produce toxin A and/or toxin B, which are important virulence factors in the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect either the bacterium or the toxins. We have developed an assay (Cdifftox activity assay) to detect C. difficile toxin A and B activities that is quantitative and cost-efficient and utilizes a substrate that is stereochemically similar to the native substrate of the toxins (UDP-glucose). To characterize toxin activity, toxins A and B were purified from culture supernatants by ammonium sulfate precipitation and chromatography through DEAE-Sepharose and gel filtration columns. The activities of the final fractions were quantitated using the Cdifftox activity assay and compared to the results of a toxin A- and B-specific enzyme-linked immunosorbent assay (ELISA). The affinity for the substrate was >4-fold higher for toxin B than for toxin A. Moreover, the rate of cleavage of the substrate was 4.3-fold higher for toxin B than for toxin A. The optimum temperature for both toxins ranged from 35 to 40°C at pH 8. Culture supernatants from clinical isolates obtained from the stools of patients suspected to be suffering from CDI were tested using the Cdifftox activity assay, and the results were compared to those of ELISA and PCR amplification of the toxin genes. Our results demonstrate that this new assay is comparable to the current commercial ELISA for detecting the toxins in the samples tested and has the added advantage of quantitating toxin activity.

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

PubMed Central

Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

2012-01-01

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates

PubMed Central

Anderson, Michele J.; Schaaf, Emily; Wallis, Heidi W.; Johnson, James R.; Tkaczyk, Christine; Sellman, Bret R.; Sun, Jisun; Peterson, Marnie L.

2018-01-01

Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections. PMID:29659477

Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model

PubMed Central

Smith, Heidi L.; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C.

2016-01-01

ABSTRACT Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16–21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38–59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans. PMID:27070129

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.

PubMed

Herrera, Cristina; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

2016-01-01

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin's enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin's bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking

PubMed Central

Herrera, Cristina; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten

2016-01-01

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells. PMID:27300140

Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

PubMed

Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

2016-08-17

Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

PubMed Central

Barrera, Daniel J.; Rosenberg, Julian N.; Chiu, Joanna G.; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T.; Shoemaker, Charles B.; Oyler, George A.; Mayfield, Stephen P.

2015-01-01

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VHH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen binding proteins and the heterodimer fusion protein containing two VHH domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism. PMID:25229405

Acetalated Dextran Microparticulate Vaccine Formulated via Coaxial Electrospray Preserves Toxin Neutralization and Enhances Murine Survival Following Inhalational Bacillus Anthracis Exposure.

PubMed

Gallovic, Matthew D; Schully, Kevin L; Bell, Matthew G; Elberson, Margaret A; Palmer, John R; Darko, Christian A; Bachelder, Eric M; Wyslouzil, Barbara E; Keane-Myers, Andrea M; Ainslie, Kristy M

2016-10-01

Subunit formulations are regarded as the safest type of vaccine, but they often contain a protein-based antigen that can result in significant challenges, such as preserving antigenicity during formulation and administration. Many studies have demonstrated that encapsulation of protein antigens in polymeric microparticles (MPs) via emulsion techniques results in total IgG antibody titers comparable to alum formulations, however, the antibodies themselves are non-neutralizing. To address this issue, a coaxial electrohydrodynamic spraying (electrospray) technique is used to formulate a microparticulate-based subunit anthrax vaccine under conditions that minimize recombinant protective antigen (rPA) exposure to harsh solvents and high shear stress. rPA and the adjuvant resiquimod are encapsulated either in separate or the same acetalated dextran MPs. Using a murine model, the electrospray formulations lead to higher IgG2a subtype titers as well as comparable total IgG antibody titers and toxin neutralization relative to the FDA-approved vaccine (BioThrax). BioThrax provides no protection against a lethal inhalational challenge of the highly virulent Ames Bacillus anthracis anthrax strain, whereas 50% of the mice vaccinated with separately encapsulated electrospray MPs survive. Overall, this study demonstrates the potential use of electrospray for encapsulating protein antigens in polymeric MPs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

PubMed Central

Gillespie, Eugene J.; Ho, Chi-Lee C.; Balaji, Kavitha; Clemens, Daniel L.; Deng, Gang; Wang, Yao E.; Elsaesser, Heidi J.; Tamilselvam, Batcha; Gargi, Amandeep; Dixon, Shandee D.; France, Bryan; Chamberlain, Brian T.; Blanke, Steven R.; Cheng, Genhong; de la Torre, Juan Carlos; Brooks, David G.; Jung, Michael E.; Colicelli, John; Damoiseaux, Robert; Bradley, Kenneth A.

2013-01-01

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease. PMID:24191014

Removal of Paralytic Shellfish Toxins by Probiotic Lactic Acid Bacteria

PubMed Central

Vasama, Mari; Kumar, Himanshu; Salminen, Seppo; Haskard, Carolyn A.

2014-01-01

Paralytic shellfish toxins (PSTs) are non-protein neurotoxins produced by saltwater dinoflagellates and freshwater cyanobacteria. The ability of Lactobacillus rhamnosus strains GG and LC-705 (in viable and non-viable forms) to remove PSTs (saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxins 2 and 3 (GTX2/3), C-toxins 1 and 2 (C1/2)) from neutral and acidic solution (pH 7.3 and 2) was examined using HPLC. Binding decreased in the order of STX ~ neoSTX > C2 > GTX3 > GTX2 > C1. Removal of STX and neoSTX (77%–97.2%) was significantly greater than removal of GTX3 and C2 (33.3%–49.7%). There were no significant differences in toxin removal capacity between viable and non-viable forms of lactobacilli, which suggested that binding rather than metabolism is the mechanism of the removal of toxins. In general, binding was not affected by the presence of other organic molecules in solution. Importantly, this is the first study to demonstrate the ability of specific probiotic lactic bacteria to remove PSTs, particularly the most toxic PST-STX, from solution. Further, these results warrant thorough screening and assessment of safe and beneficial microbes for their usefulness in the seafood and water industries and their effectiveness in vivo. PMID:25046082

Human intravenous immunoglobulin for experimental streptococcal toxic shock: bacterial clearance and modulation of inflammation.

PubMed

Sriskandan, Shiranee; Ferguson, Melissa; Elliot, Victoria; Faulkner, Lee; Cohen, Jonathan

2006-07-01

Polyclonal human intravenous immunoglobulin (IVIG) has been advocated as an adjunct to therapy in severe invasive streptococcal toxic shock because of its ability to neutralize superantigen toxins. The aim of this study was to assess IVIG therapeutic efficacy in an experimental model of streptococcal toxic shock. To confirm the in vitro activity of IVIG against the Streptococcus pyogenes strain used in the study, IVIG was tested for superantigen neutralizing and bacterial opsonizing activity prior to in vivo studies. To evaluate the in vivo effects of IVIG in terms of microbiological outcome and disease severity in a superantigen-sensitive transgenic model of streptococcal shock, HLA-DQ transgenic mice were treated with IVIG either at the time of infection or after infection with S. pyogenes. Antibiotics were included in some studies. The IVIG preparation neutralized superantigenicity of S. pyogenes in vitro and enhanced bacterial killing in a whole blood assay. When given to mice at the time of S. pyogenes infection, IVIG neutralized circulating superantigens and reduced systemic inflammatory response. Remarkably, IVIG-enhanced systemic clearance of bacteria and enhanced neutrophil infiltrate into the infected tissues. However, when used in combination with penicillin and clindamycin in a delayed treatment setting, IVIG did not confer additional therapeutic benefit, in terms of inflammatory response, bacterial clearance or survival. IVIG monotherapy can confer benefit in experimental streptococcal shock, but extension of these findings to the clinical situation will require further evaluation.

Detection of Clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia.

PubMed

Perkins, S E; Fox, J G; Taylor, N S; Green, D L; Lipman, N S

1995-08-01

Four specific-pathogen-free rabbits with anorexia died peracutely; decreased fecal output, nasal exudate, and labored breathing were the only other clinical abnormalities observed in two of the rabbits before death. The animals, three juveniles and one adult, were on a standard polyclonal antibody production regimen and had received immunizations approximately 2 weeks before presentation. External examination revealed distended abdomen and perineal fecal staining. At necropsy the small intestine was distended with fluid, and the cecum was distended with chyme. The small intestines and cecum had marked serosal hyperemia. Anaerobic bacterial culture techniques were used to isolate Clostridium difficile from the small intestine (3/4) and cecum (2/4). In all cases C. difficile toxin B was detected at high titers (10(2) to > 10(5)) in the small intestine by cytotoxicity assay with HeLa 229 cell culture. In two of the four rabbits C. difficile was isolated, and cytotoxin titers were detected at 10(1) and 10(4) in the cecum of affected rabbits. Toxin B was neutralized with C. sordellii antiserum but not C. spiroforme antiserum. In addition, toxin A was detected in each of the cytotoxin B-positive samples by a commercial toxin A enzyme immunosorbent assay. In vitro production of toxins A and B was detected from each culture isolate after incubation in chopped meat broth. These cases are noteworthy because spontaneous (nonantibiotic-associated) C. difficile enterotoxemia has not been previously reported in rabbits. Also the toxins of clostridial organisms are usually documented in the cecum, not the small intestine, of rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

PubMed Central

Monti, Maria C.; Hernández-Arriaga, Ana M.; Kamphuis, Monique B.; López-Villarejo, Juan; Heck, Albert J. R.; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H. H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon. PMID:17317682

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea

PubMed Central

Sack, David A.

2015-01-01

Diarrhea continues to be a leading cause of death in children <5 years of age, and enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of children's diarrhea. Currently, there are no available vaccines against ETEC-associated diarrhea. Whole-cell vaccine candidates have been under development but require further improvements because they provide inadequate protection and produce unwanted adverse effects. Meanwhile, a newer approach using polypeptide or subunit vaccine candidates focusing on ETEC colonization factor antigens (CFAs) and enterotoxins, the major virulence determinants of ETEC diarrhea, shows substantial promise. A conservative CFA/I adhesin tip antigen and a CFA MEFA (multiepitope fusion antigen) were shown to induce cross-reactive antiadhesin antibodies that protected against adherence by multiple important CFAs. Genetic fusion of toxoids derived from ETEC heat-labile toxin (LT) and heat-stable toxin (STa) induced antibodies neutralizing both enterotoxins. Moreover, CFA-toxoid MEFA polypeptides, generated by fusing CFA MEFA to an STa-LT toxoid fusion, induced antiadhesin antibodies that broadly inhibited adherence of the seven most important ETEC CFAs associated with about 80% of the diarrhea cases caused by ETEC strains with known CFAs. This same antigen preparation also induced antitoxin antibodies that neutralized both toxins that are associated with all cases of ETEC diarrhea. Results from these studies suggest that polypeptide or subunit vaccines have the potential to effectively protect against ETEC diarrhea. In addition, novel adhesins and mucin proteases have been investigated as potential alternatives or, more likely, additional antigens for ETEC subunit vaccine development. PMID:26135975

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

PubMed

Martorelli, Luisina; Garbaccio, Sergio; Vilte, Daniel A; Albanese, Adriana A; Mejías, María P; Palermo, Marina S; Mercado, Elsa C; Ibarra, Cristina E; Cataldi, Angel A

2017-01-01

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

Purification and Characterization of a Novel Subtype A3 Botulinum Neurotoxin

PubMed Central

Tepp, William H.; Lin, Guangyun

2012-01-01

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 × 107 50% lethal doses (LD50)/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins. PMID:22367089

Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE.

PubMed

Kamphuis, Monique B; Monti, Maria Chiara; van den Heuvel, Robert H H; Santos-Sierra, Sandra; Folkers, Gert E; Lemonnier, Marc; Díaz-Orejas, Ramón; Heck, Albert J R; Boelens, Rolf

2007-04-01

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed. (c) 2007 Wiley-Liss, Inc.

Purification and characterization of a novel subtype a3 botulinum neurotoxin.

PubMed

Tepp, William H; Lin, Guangyun; Johnson, Eric A

2012-05-01

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 × 10(7) 50% lethal doses (LD(50))/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins.

Sensitivity of mouse bioassay in clinical wound botulism.

PubMed

Wheeler, Charlotte; Inami, Gregory; Mohle-Boetani, Janet; Vugia, Duc

2009-06-15

California has an ongoing epidemic of wound botulism (WB) among injection drug users (IDUs). We retrospectively studied a cohort of patients with WB and determined the sensitivity of the mouse bioassay-the gold standard laboratory test for confirmation of botulism-in verifying WB. We defined a clinical case of WB as an acute, bilateral, descending, flaccid paralysis starting with 1 cranial nerve palsies in an IDU with no other explainable diagnosis. We calculated the sensitivity of the mouse bioassay as the proportion of clinical WB cases that had positive serum toxin test results by mouse bioassay. We compared serum toxin-positive with serum toxin-negative patients. Of 73 patients with WB, 50 tested serum toxin positive, yielding a sensitivity of 68%. Serum toxin-positive patients did not differ significantly from serum toxin-negative patients with respect to demographic characteristics or injection drug use practices or in days from patient symptom onset to collection of specimens for testing. Patients did not differ significantly by clinical characteristics, except that serum toxin-positive patients were more likely than serum toxin-negative patients to have required mechanical ventilation during their hospital courses (74% vs. 43%; P = .01). In this study, the mouse bioassay failed to detect botulinum toxin in the serum samples of nearly one-third of IDUs with characteristic WB. Such patients should be considered to have probable WB. Physicians should be aware of the test's limitations and base their final diagnosis of suspected WB on clinical criteria when the mouse bioassay produces negative results.

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine.

PubMed

Phaswana, P H; Ndumnego, O C; Koehler, S M; Beyer, W; Crafford, J E; van Heerden, H

2017-09-07

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (r s  ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

PubMed

Karau, Melissa J; Tilahun, Mulualem E; Krogman, Ashton; Osborne, Barbara A; Goldsby, Richard A; David, Chella S; Mandrekar, Jayawant N; Patel, Robin; Rajagopalan, Govindarajan

2017-10-03

Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

In-Vivo Neutralization of Botulinum Neurotoxin Serotype E Using Rabbit Polyclonal Antibody Developed against BoNT/E Light Chain.

PubMed

Rani, Sarita; Ponmariappan, S; Sharma, Arti; Kamboj, D V; Jain, A K

2017-01-01

Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment. In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours. The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F. The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition.

PubMed

Arana, Angela M; Bierdeman, Michael A; Balzli, Charles L; Tang, Aihua; Caballero, Armando R; Patel, Rupesh; O'Callaghan, Richard J

2015-01-01

Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents. The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-β-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis. At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019). Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.

PubMed

Slayden, Richard A; Dawson, Clinton C; Cummings, Jason E

2018-06-01

There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

Stabilization of a Recombinant Ricin Toxin A Subunit Vaccine through Lyophilization

PubMed Central

Hassett, Kimberly J.; Cousins, Megan C.; Rabia, Lilia A.; Chadwick, Chrystal M.; O’Hara, Joanne M.; Nandi, Pradyot; Brey, Robert N.; Mantis, Nicholas J.; Carpenter, John F.; Randolph, Theodore W.

2013-01-01

Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40°C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNA) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40°C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40°C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge. PMID:23583494

Stabilization of a recombinant ricin toxin A subunit vaccine through lyophilization.

PubMed

Hassett, Kimberly J; Cousins, Megan C; Rabia, Lilia A; Chadwick, Chrystal M; O'Hara, Joanne M; Nandi, Pradyot; Brey, Robert N; Mantis, Nicholas J; Carpenter, John F; Randolph, Theodore W

2013-10-01

Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40 °C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNAs) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40 °C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40 °C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge. Copyright © 2013 Elsevier B.V. All rights reserved.

Excitable toxin-antitoxin modules coordinated through intracellular bottlenecks

NASA Astrophysics Data System (ADS)

Mather, William

Chronic infections and pathogenic biofilms present a serious threat to the health of humans by decreasing life expectancy and quality. The resilience of these microbial communities has been attributed to the spontaneous formation of persister cells, which constitute a small fraction of the population capable of surviving a wide range of environmental stressors. Gating of bacterial persistence has recently been linked to toxin-antitoxin (TA) modules, which are operons with an evolutionarily conserved motif that includes a toxin that halts cell growth and a corresponding antitoxin that neutralizes the toxin. While many such modules have been identified and studied in a wide range of organisms, little consideration of the interactions between multiple modules within a single host has been made. Moreover, the multitude of different antitoxin species are degraded by a relatively small number of proteolytic pathways, strongly suggesting competition between antitoxins for degradation machinery, i.e. queueing coupling. Here we present a theoretical understanding of the dynamics of multiple TA modules that are coupled through either proteolytic queueing, a toxic effect on cell growth rate, or both. We conclude that indirect queueing coordination between multiple TA modules may be central to controlling bacterial persistence. NSF Award Number MCB-1330180.

The ClpXP protease is responsible for the degradation of the Epsilon antidote to the Zeta toxin of the streptococcal pSM19035 plasmid.

PubMed

Brzozowska, Iwona; Zielenkiewicz, Urszula

2014-03-14

Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ε-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ε/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ε-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His6-Epsilon by the His6-LonBs, ClpPBs, and ClpXBs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.

Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection?

PubMed

Garvey, Mark I; Bradley, Craig W; Wilkinson, Martyn A C; Holden, Elisabeth

2017-01-01

Diagnosis of C. difficile infection (CDI) is controversial because of the many laboratory methods available and their lack of ability to distinguish between carriage, mild or severe disease. Here we describe whether a low C. difficile toxin B nucleic acid amplification test (NAAT) cycle threshold (CT) can predict toxin EIA, CDI severity and mortality. A three-stage algorithm was employed for CDI testing, comprising a screening test for glutamate dehydrogenase (GDH), followed by a NAAT, then a toxin enzyme immunoassay (EIA). All diarrhoeal samples positive for GDH and NAAT between 2012 and 2016 were analysed. The performance of the NAAT CT value as a classifier of toxin EIA outcome was analysed using a ROC curve; patient mortality was compared to CTs and toxin EIA via linear regression models. A CT value ≤26 was associated with ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. A CT value of ≤26 was significantly associated ( p  = 0.0262) with increased one month mortality, severe cases of CDI or failure of first line treatment. The ROC curve probabilities demonstrated a CT cut off value of 26.6. Here we demonstrate that a CT ≤26 indicates more severe CDI and is associated with higher mortality. Samples with a low CT value are often toxin EIA positive, questioning the need for this additional EIA test. A CT ≤26 could be used to assess the potential for severity of CDI and guide patient treatment.

Neutralization of toxicological activities of medically-relevant Bothrops snake venoms and relevant toxins by two polyvalent bothropic antivenoms produced in Peru and Brazil.

PubMed

Estevao-Costa, Maria I; Gontijo, Silea S; Correia, Barbara L; Yarleque, Armando; Vivas-Ruiz, Dan; Rodrigues, Edith; Chávez-Olortegui, Carlos; Oliveira, Luciana S; Sanchez, Eladio F

2016-11-01

Snakebite envenoming is a neglected public pathology, affecting especially rural communities or isolated areas of tropical and subtropical Latin American countries. The parenteral administration of antivenom is the mainstay and the only validated treatment of snake bite envenoming. Here, we assess the efficacy of polyspecific anti-Bothrops serum (α-BS) produced in the Instituto Nacional de Salud (INS, Peru) and at the Fundação Ezequiel Dias (FUNED, Brazil), to neutralize the main toxic activities induced by five medically-relevant venoms of: Bothrops atrox, B. barnetti, and B. pictus from Peru, and the Brazilian B. jararaca and B. leucurus, all of them inhabiting different geographical locations. Protein electrophoretic patterns of these venoms showed significant differences in composition, number and intensity of bands. Another goal was to evaluate the efficacy and safety of lyophilized α-BS developed at INS to neutralize the detrimental effects of these venoms using in vivo and in vitro assays. The availability of lyophilized α-BS has relevant significance in its distribution to distant rural communities where the access to antivenom in health facilities is more difficult. Despite the fact that different antigen mixtures were used for immunization during antivenom production, our data showed high toxin-neutralizing activity of α-BS raised against Bothrops venoms. Moreover, the antivenom cross-reacted even against venoms not included in the immunization mixture. Furthermore, we have evaluated the efficacy of both α-BS to neutralize key toxic compounds belonging to the predominant protein families of Bothrops snakes. Most significantly, both α-BS cross-specifically neutralized the main toxicological activities e.g. lethality and hemorrhage induced by these venoms. Thus, our data indicate that both α-BS are equally effective to treat snake bite victims inflicted by Bothrops snakes particularly B. atrox, responsible for the largest numbers of human envenomations in the Amazon regions of some South American countries including Peru and Brazil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

PubMed Central

Kim, Younghoon; Arruda, Jennifer M.; Davenport, Andrew; Wood, Thomas K.; Peti, Wolfgang; Page, Rebecca

2009-01-01

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state. PMID:20041169

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus.

PubMed

Hino, M; Zhang, J; Takagi, H; Miyoshi, T; Uchiumi, T; Nakashima, T; Kakuta, Y; Kimura, M

2014-07-01

To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division. © 2014 The Society for Applied Microbiology.

Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

PubMed

Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

2016-12-15

The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs

USGS Publications Warehouse

Liao, Chih-Ming; Huang, Chienjin; Hsuan, Shih-Ling; Chen, Zeng-Weng; Lee, Wei-Cheng; Liu, Cheng-I; Winton, James R.; Chien, Maw-Sheng

2006-01-01

Three short fragments of recombinant subunit Pasteurella multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. PMT-specific antibody secreting cells and evidence of cellular immunity were detected in rsPMT-immunized pigs following authentic PMT challenge or homologous antigen booster. Piglets immunized with rsPMT fragments containing either the N-terminal or the C-terminal portions of PMT developed high titers of neutralizing antibodies. Pregnant sows immunized with rsPMT had higher levels of maternal antibodies in their colostrum than did those immunized with a conventional PAR-toxoid vaccine. Offspring from rsPMT vaccinated sows had better survival after challenge with a five-fold lethal dose of authentic PMT and had better growth performance after challenge with a sublethal dose of toxin. Our findings indicate these non-toxic rsPMT proteins are attractive candidates for development of a subunit vaccine against PAR in pigs.

Elimination of bioweapons agents from forensic samples during extraction of human DNA.

PubMed

Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

2014-11-01

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling. © 2014 American Academy of Forensic Sciences.

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.

PubMed

Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F M; Oliveira, Daysiane; Machado de Avila, Ricardo A; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S; Minozzo, João C; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

2018-01-01

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho , and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

PubMed Central

Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F. M.; Oliveira, Daysiane; Machado de Avila, Ricardo A.; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S.; Minozzo, João C.; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

2018-01-01

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms. PMID:29666624

Clostridium difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms ▿

PubMed Central

Novak-Weekley, Susan M.; Marlowe, Elizabeth M.; Miller, John M.; Cumpio, Joven; Nomura, Jim H.; Vance, Paula H.; Weissfeld, Alice

2010-01-01

The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study. PMID:20071552

Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

PubMed Central

Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

2014-01-01

Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

Type F botulism due to neurotoxigenic Clostridium baratii from an unknown source in an adult.

PubMed

McCroskey, L M; Hatheway, C L; Woodruff, B A; Greenberg, J A; Jurgenson, P

1991-11-01

Type F botulism was confirmed in a 54-year-old male with signs compatible with botulism who reported to the emergency unit of a hospital. Botulinal neurotoxin was detected in the patient's serum and fecal specimens, and a neurotoxigenic organism whose physiologic characteristics correspond to those of Clostridium baratii was isolated. The toxin produced by the isolate was neutralized by type F botulinal antitoxin and cross-neutralized with lower efficiency by type E antitoxin. The patient's food history was not suggestive of botulism, and it seems likely that the illness was due to colonization of the gut.

Type F botulism due to neurotoxigenic Clostridium baratii from an unknown source in an adult.

PubMed Central

McCroskey, L M; Hatheway, C L; Woodruff, B A; Greenberg, J A; Jurgenson, P

1991-01-01

Type F botulism was confirmed in a 54-year-old male with signs compatible with botulism who reported to the emergency unit of a hospital. Botulinal neurotoxin was detected in the patient's serum and fecal specimens, and a neurotoxigenic organism whose physiologic characteristics correspond to those of Clostridium baratii was isolated. The toxin produced by the isolate was neutralized by type F botulinal antitoxin and cross-neutralized with lower efficiency by type E antitoxin. The patient's food history was not suggestive of botulism, and it seems likely that the illness was due to colonization of the gut. PMID:1774272

Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits.

PubMed

Roetzer, Andreas; Gruener, Corina S; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M

2016-10-28

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SE l N, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SE l N led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSE l N led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens.

Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits

PubMed Central

Roetzer, Andreas; Gruener, Corina S.; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M.

2016-01-01

Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SElN, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SElN led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSElN led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens. PMID:27801832

Clostridium difficile infection diagnostics - evaluation of the C. DIFF Quik Chek Complete assay, a rapid enzyme immunoassay for detection of toxigenic C. difficile in clinical stool samples.

PubMed

Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn

2016-11-01

Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Venom of the Coral Snake Micrurus clarki: Proteomic Profile, Toxicity, Immunological Cross-Neutralization, and Characterization of a Three-Finger Toxin.

PubMed

Lomonte, Bruno; Sasa, Mahmood; Rey-Suárez, Paola; Bryan, Wendy; Gutiérrez, José María

2016-05-05

Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a 'venomics' approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A₂ (PLA₂; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, 'intermediate' type within the dichotomy between 3FTx- and PLA₂-predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA₂ venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema.

The development of IgY(DeltaFc) antibody based neuro toxin antivenoms and the study on their neutralization efficacies.

PubMed

Chiou, Victor Y-Neng

2008-07-01

Immunotherapy for treatment of snake bites has been based on mammalian IgG. Recently, polyvalent ovine Fab has become available. However, papain, used in the Fab fragmentation process, is a human allergen. Avian eggs are a source of antibodies and a truncated version of IgY, IgY(DeltaFc), is found in ducks. In this study, we induced duck antibodies by using detoxified cobra and krait venoms and then purified IgY(DeltaFc) antibodies from the hyperimmune duck egg yolk. Ducks were used for immunization and their eggs were collected for antibody production. ICR strain female mice were used in the in vivo neutralization test. Monovalent antivenoms to Formosan cobra venom and Formosan multi-banded krait venom were raised and purified from hyper-immune duck egg yolk individually. The LD(50) of venoms were determined by subcutaneous injection of different venom doses into the mice. The survival/death ratios were recorded after 24 hours. The antibody purified from egg yolk showed high titer response to its immunogen (cobra or krait venom) by an ELISA. Overall, the antibodies from duck eggs efficiently protected mice from envenomations. The antivenoms purified from the egg yolk of ducks immunized with cobra venom and krait venom neutralized the lethal effects of these venoms with good efficacy in a mouse model. The antivenoms were effective in neutralizing lethality in mice injected at 4xLD(50) of venoms. These results indicate that antibodies derived from ducks can serve as a new source for the generation of antivenoms.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides

PubMed Central

Wiertsema, Selma P.; Corscadden, Karli J.; Mateus, Tulia; Mullaney, Gemma L.; Zhang, Guicheng; Richmond, Peter C.; Thornton, Ruth B.

2016-01-01

ABSTRACT The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. PMID:28031178

Venom of Bothrops asper from Mexico and Costa Rica: intraspecific variation and cross-neutralization by antivenoms.

PubMed

Segura, Alvaro; Herrera, María; Villalta, Mauren; Vargas, Mariángela; Uscanga-Reynell, Alfredo; de León-Rosales, Samuel Ponce; Jiménez-Corona, María Eugenia; Reta-Mares, José Francisco; Gutiérrez, José María; León, Guillermo

2012-01-01

Bothrops asper is the species that induces the highest incidence of snakebite envenomation in southern Mexico, Central America and parts of northern South America. The intraspecies variability in HPLC profile and toxicological activities between the venoms from specimens collected in Mexico (Veracruz) and Costa Rica (Caribbean and Pacific populations) was investigated, as well as the cross-neutralization by antivenoms manufactured in these countries. Venoms differ in their HPLC profiles and in their toxicity, since venom from Mexican population showed higher lethal and defibrinogenating activities, whereas those from Costa Rica showed higher hemorrhagic and in vitro coagulant activities. In general, antivenoms were more effective in the neutralization of homologous venoms. Overall, both antivenoms effectively neutralized the various toxic effects of venoms from the two populations of B. asper. However, antivenom raised against venom from Costa Rican specimens showed a higher efficacy in the neutralization of defibrinogenating and coagulant activities, thus highlighting immunochemical differences in the toxins responsible for these effects associated with hemostatic disturbances in snakebite envenoming. These observations illustrate how intraspecies venom variation may influence antivenom neutralizing profile. Copyright © 2011 Elsevier Ltd. All rights reserved.

Anchoring antibodies to membranes using a diphtheria toxin T domain-ZZ fusion protein as a pH sensitive membrane anchor.

PubMed

Nizard, P; Liger, D; Gaillard, C; Gillet, D

1998-08-14

We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

Molecular cloning and characterization of Bacillus alvei thiol-dependent cytolytic toxin expressed in Escherichia coli.

PubMed

Geoffroy, C; Alouf, J E

1988-07-01

A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.

Ricin vaccine development.

PubMed

Smallshaw, Joan E; Vitetta, Ellen S

2012-01-01

In this chapter we discuss vaccines to protect against the highly toxic plant-derived toxin, ricin. Due to its prevalence, ease of use, and stability it has been used in sporadic incidents of espionage. There is also concern that it will be used as an agent of bioterrorism. As a result there has been a great deal of interest in developing a safe vaccine or antidote to protect humans, and in particular soldiers and first responders. Although multiple types of vaccines have been tested, at this time two recombinant vaccines are the leading candidates for the national vaccine stockpile. In terms of passive post-exposure protection, monoclonal neutralizing antibodies that passively protect animals are also under development. These vaccines and antibodies are discussed in the context of the toxicity and structure of ricin.

Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile.

PubMed

Qutub, M O; AlBaz, N; Hawken, P; Anoos, A

2011-01-01

To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer's instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.

A two-stage algorithm for Clostridium difficile including PCR: can we replace the toxin EIA?

PubMed

Orendi, J M; Monnery, D J; Manzoor, S; Hawkey, P M

2012-01-01

A two step, three-test algorithm for Clostridium difficile infection (CDI) was reviewed. Stool samples were tested by enzyme immunoassays for C. difficile common antigen glutamate dehydrogenase (G) and toxin A/B (T). Samples with discordant results were tested by polymerase chain reaction detecting the toxin B gene (P). The algorithm quickly identified patients with detectable toxin A/B, whereas a large group of patients excreting toxigenic C. difficile but with toxin A/B production below detection level (G(+)T(-)P(+)) was identified separately. The average white blood cell count in patients with a G(+)T(+) result was higher than in those with a G(+)T(-)P(+) result. Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Establishment of alternative potency test for botulinum toxin type A using compound muscle action potential (CMAP) in rats.

PubMed

Torii, Yasushi; Goto, Yoshitaka; Nakahira, Shinji; Ginnaga, Akihiro

2014-11-01

The biological activity of botulinum toxin type A has been evaluated using the mouse intraperitoneal (ip) LD50 test. This method requires a large number of mice to precisely determine toxin activity, and, as such, poses problems with regard to animal welfare. We previously developed a compound muscle action potential (CMAP) assay using rats as an alternative method to the mouse ip LD50 test. In this study, to evaluate this quantitative method of measuring toxin activity using CMAP, we assessed the parameters necessary for quantitative tests according to ICH Q2 (R1). This assay could be used to evaluate the activity of the toxin, even when inactive toxin was mixed with the sample. To reduce the number of animals needed, this assay was set to measure two samples per animal. Linearity was detected over a range of 0.1-12.8 U/mL, and the measurement range was set at 0.4-6.4 U/mL. The results for accuracy and precision showed low variability. The body weight was selected as a variable factor, but it showed no effect on the CMAP amplitude. In this study, potency tests using the rat CMAP assay of botulinum toxin type A demonstrated that it met the criteria for a quantitative analysis method. Copyright © 2014 Elsevier Ltd. All rights reserved.

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era.

PubMed

Polage, Christopher R; Gyorke, Clare E; Kennedy, Michael A; Leslie, Jhansi L; Chin, David L; Wang, Susan; Nguyen, Hien H; Huang, Bin; Tang, Yi-Wei; Lee, Lenora W; Kim, Kyoungmi; Taylor, Sandra; Romano, Patrick S; Panacek, Edward A; Goodell, Parker B; Solnick, Jay V; Cohen, Stuart H

2015-11-01

Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.

Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients.

PubMed

Erb, S; Frei, R; Strandén, A M; Dangel, M; Tschudin-Sutter, S; Widmer, A F

2015-11-01

The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12,481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/μL (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Glucosylation and Other Biotransformations of T-2 Toxin by Yeasts of the Trichomonascus Clade

PubMed Central

Price, Neil P. J.; Kurtzman, Cletus P.

2012-01-01

Trichothecenes are sesquiterpenoid toxins produced by Fusarium species. Since these mycotoxins are very stable, there is interest in microbial transformations that can remove toxins from contaminated grain or cereal products. Twenty-three yeast species assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including four Trichomonascus species and 19 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert the trichothecene T-2 toxin to less-toxic products. These species gave three types of biotransformations: acetylation to 3-acetyl T-2 toxin, glycosylation to T-2 toxin 3-glucoside, and removal of the isovaleryl group to form neosolaniol. Some species gave more than one type of biotransformation. Three Blastobotrys species converted T-2 toxin into T-2 toxin 3-glucoside, a compound that has been identified as a masked mycotoxin in Fusarium-infected grain. This is the first report of a microbial whole-cell method for producing trichothecene glycosides, and the potential large-scale availability of T-2 toxin 3-glucoside will facilitate toxicity testing and development of methods for detection of this compound in agricultural and other products. PMID:23042183

A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a Complete tcdA Gene

PubMed Central

Marín, Mercedes; Martín, Adoración; Rupnik, Maja

2014-01-01

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A− B+ CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site. PMID:25428159

Residual Limb Hyperhidrosis Managed by Botulinum Toxin Injections, Enhanced by the Iodine-Starch Test: A Case Report.

PubMed

Hansen, Colby R; Godfrey, Bradeigh

2017-04-01

Hyperhidrosis of the residual limb is a common condition affecting patients with amputations. The iodine-starch test is used by dermatologists to identify focal areas of hyperhidrosis before treatment with botulinum toxin. Here, we describe a case of a patient with a transtibial amputation with moderate-to-severe hyperhidrosis who received intradermal botulinum toxin injections to treat residual limb hyperhidrosis, with particular emphasis given to the utility of the iodine-starch test in managing this common condition. The iodine-starch test successfully identified hyperhidrotic areas before treatment as well as confirmed the physiologic anhidrotic effect of the botulinum toxin treatment. V. Copyright © 2017 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

PubMed Central

2012-01-01

Background Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized “Photorhabdus virulence cassettes (PVC)”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative ‘cheating’ in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers. Reviewers This article was reviewed by AM, FE and IZ. PMID:22731697

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7

PubMed Central

Martorelli, Luisina; Garbaccio, Sergio; Vilte, Daniel A.; Albanese, Adriana A.; Mejías, María P.; Palermo, Marina S.; Mercado, Elsa C.; Ibarra, Cristina E.; Cataldi, Angel A.

2017-01-01

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo. PMID:28046078

Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water

PubMed Central

Jardon-Xicotencatl, Samantha; Díaz-Torres, Roberto; Marroquín-Cardona, Alicia; Villarreal-Barajas, Tania; Méndez-Albores, Abraham

2015-01-01

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize. PMID:26512692

[New methodological advances: algorithm proposal for management of Clostridium difficile infection].

PubMed

González-Abad, María José; Alonso-Sanz, Mercedes

2015-06-01

Clostridium difficile infection (CDI) is considered the most common cause of health care-associated diarrhea and also is an etiologic agent of community diarrhea. The aim of this study was to assess the potential benefit of a test that detects glutamate dehydrogenase (GDH) antigen and C. difficile toxin A/B, simultaneously, followed by detection of C. difficile toxin B (tcdB) gene by PCR as confirmatory assay on discrepant samples, and to propose an algorithm more efficient. From June 2012 to January 2013 at Hospital Infantil Universitario Niño Jesús, Madrid, the stool samples were studied for the simultaneous detection of GDH and toxin A/B, and also for detection of toxin A/B alone. When results between GDH and toxin A/B were discordant, a single sample for patient was selected for detection of C. difficile toxin B (tcdB) gene. A total of 116 samples (52 patients) were tested. Four were positive and 75 negative for toxigenic C. difficile (Toxin A/B, alone or combined with GDH). C. difficile was detected in the remaining 37 samples but not toxin A/B, regardless of the method used, except one. Twenty of the 37 specimens were further tested for C. difficile toxin B (tcdB) gene and 7 were positive. The simultaneous detection of GDH and toxin A/B combined with PCR recovered undiagnosed cases of CDI. In accordance with our data, we propose a two-step algorithm: detection of GDH and PCR (in samples GDH positive). This algorithm could provide a superior cost-benefit ratio in our population.

Genome-Wide Association Studies of Metabolites in Patients with CKD Identify Multiple Loci and Illuminate Tubular Transport Mechanisms.

PubMed

Li, Yong; Sekula, Peggy; Wuttke, Matthias; Wahrheit, Judith; Hausknecht, Birgit; Schultheiss, Ulla T; Gronwald, Wolfram; Schlosser, Pascal; Tucci, Sara; Ekici, Arif B; Spiekerkoetter, Ute; Kronenberg, Florian; Eckardt, Kai-Uwe; Oefner, Peter J; Köttgen, Anna

2018-05-01

Background The kidneys have a central role in the generation, turnover, transport, and excretion of metabolites, and these functions can be altered in CKD. Genetic studies of metabolite concentrations can identify proteins performing these functions. Methods We conducted genome-wide association studies and aggregate rare variant tests of the concentrations of 139 serum metabolites and 41 urine metabolites, as well as their pairwise ratios and fractional excretions in up to 1168 patients with CKD. Results After correction for multiple testing, genome-wide significant associations were detected for 25 serum metabolites, two urine metabolites, and 259 serum and 14 urinary metabolite ratios. These included associations already known from population-based studies. Additional findings included an association for the uremic toxin putrescine and variants upstream of an enzyme catalyzing the oxidative deamination of polyamines ( AOC1 , P -min=2.4×10 -12 ), a relatively high carrier frequency (2%) for rare deleterious missense variants in ACADM that are collectively associated with serum ratios of medium-chain acylcarnitines ( P -burden=6.6×10 -16 ), and associations of a common variant in SLC7A9 with several ratios of lysine to neutral amino acids in urine, including the lysine/glutamine ratio ( P =2.2×10 -23 ). The associations of this SLC7A9 variant with ratios of lysine to specific neutral amino acids were much stronger than the association with lysine concentration alone. This finding is consistent with SLC7A9 functioning as an exchanger of urinary cationic amino acids against specific intracellular neutral amino acids at the apical membrane of proximal tubular cells. Conclusions Metabolomic indices of specific kidney functions in genetic studies may provide insight into human renal physiology. Copyright © 2018 by the American Society of Nephrology.

Toxins and antimicrobial peptides: interactions with membranes

NASA Astrophysics Data System (ADS)

Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

2009-08-01

The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE PAGES

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

2015-03-09

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

PubMed Central

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

2015-01-01

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

Prevalence of Clostridium perfringens toxin in patients suspected of having antibiotic-associated diarrhea.

PubMed

Kim, Young Jin; Kim, Si Hyun; Ahn, Junggu; Cho, Soongmoon; Kim, Dongchun; Kim, Kwanghyun; Lee, Heegun; Son, Hyunwoo; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kim, Hye Ran; Shin, Jeong Hwan

2017-12-01

Although Clostridium perfringens has been reported as a cause of antibiotic-associated diarrhea (AAD), it is uncommon to detect this pathogen in clinical microbiology laboratories in Korea. The aim of this study was to investigate the prevalence of C. perfringens toxin in patients suspected of having AAD. A total of 135 stool specimens submitted to a clinical microbiology laboratory for C. difficile toxin assay were tested. We tried to detect both C. difficile and C. perfringens toxins using the Seeplex Diarrhea ACE Detection kit (Seegene, Seoul, Korea). We evaluated the prevalence of 10 bacteria and 5 viruses. A total of 40 Clostridium spp. were detected in 34 specimens (29.6%). The C. perfringens toxin was detected in 14 of 135 specimens (10.4%), while C. difficile toxin was detected in 26 specimens (19.3%). Other bacteria and viruses, including 8 Aeromonas spp., were detected in 15 specimens. All tests were negative in 92 of the 135 specimens (68.1%). Clostridium perfringens toxin is relatively common, and we should consider the possibility of its presence in patients suspected of having AAD, especially if C. difficile tests are negative. Copyright © 2017 Elsevier Ltd. All rights reserved.

Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows

USGS Publications Warehouse

Moeller, R.B.; Puschner, B.; Walker, R.L.; Rocke, T.E.; Smith, S.R.; Cullor, J.S.; Ardans, A.A.

2009-01-01

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood–milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.

Comparison of the neurotoxic and myotoxic effects of two Moroccan scorpion venoms and their neutralization by experimental polyclonal antivenom.

PubMed

Oukkache, Naoual; Ahmad Rusmili, Muhamad Rusdi; Othman, Iekhsan; Ghalim, Noreddine; Chgoury, Fatima; Boussadda, Lofti; Elmdaghri, Naima; Sabatier, Jean-Marc

2015-03-01

Scorpion venoms contain complex mixtures of molecules, including peptides. These peptides specifically bind to various targets, in particular ion channels. Toxins modulating Na(+), K(+), Ca(2+) and Cl(-) currents were described from venoms. The Androctonus and Buthus geni of scorpions are widely distributed in Morocco. Their stings can cause pain, inflammation, necrosis, muscle paralysis and death. The myotoxicity is predominantly associated with neurotoxic effects and is a cause of mortality and morbidity. In this study, pharmacological effects of venoms were investigated in vitro on neuromuscular transmission. Effects of Androctonus mauretanicus (Am) and Buthus occitanus (Bo) venoms were investigated using the chick biventer cervicis nerve-muscle preparations. The protective activity of antivenom was also investigated. The antivenom was made from serum of horse that was hyperimmunized with Bo and Androctonus australis hector (Aah) venoms and one venom from Middle East species (Lq). The protective activity of the antivenom was assessed on the neuromuscular system by using stimulated chick nerve-muscle. The results were compared with lethal activity neutralization in mice. Am and Bo venoms contain myotoxins and postsynaptic neurotoxins. In agreement with lethal potencies of these venoms in mice, Am venom displays greater neurotoxicity and myotoxicity. The antivenom prevented lethality caused by Am, Bo and Aah venoms. The antivenom did not prevent toxic effects caused by Am venom whereas it neutralized Bo venom. Am and Bo venoms contain distinct toxins that are responsible for myotoxicity and neurotoxicity. It would be appropriate to add Am venom to produce more efficient antivenom. Copyright © 2015 Elsevier Inc. All rights reserved.

Potentiation of an anthrax DNA vaccine with electroporation.

PubMed

Luxembourg, A; Hannaman, D; Nolan, E; Ellefsen, B; Nakamura, G; Chau, L; Tellez, O; Little, S; Bernard, R

2008-09-19

DNA vaccines are a promising method of immunization against biothreats and emerging infections because they are relatively easy to design, manufacture, store and distribute. However, immunization with DNA vaccines using conventional delivery methods often fails to induce consistent, robust immune responses, especially in species larger than the mouse. Intramuscular (i.m.) delivery of a plasmid encoding anthrax toxin protective antigen (PA) using electroporation (EP), a potent DNA delivery method, rapidly induced anti-PA IgG and toxin neutralizing antibodies within 2 weeks following a single immunization in multiple experimental species. The delivery procedure is particularly dose efficient and thus favorable for achieving target levels of response following vaccine administration in humans. These results suggest that EP may be a valuable platform technology for the delivery of DNA vaccines against anthrax and other biothreat agents.

Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from Streptococcus mitis▿

PubMed Central

Jefferies, Johanna; Nieminen, Leena; Kirkham, Lea-Ann; Johnston, Calum; Smith, Andrew; Mitchell, Tim J.

2007-01-01

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin. PMID:17071760

Immunochromatographic Strip Test for Rapid Detection of Diphtheria Toxin: Description and Multicenter Evaluation in Areas of Low and High Prevalence of Diphtheria

PubMed Central

Engler, K. H.; Efstratiou, A.; Norn, D.; Kozlov, R. S.; Selga, I.; Glushkevich, T. G.; Tam, M.; Melnikov, V. G.; Mazurova, I. K.; Kim, V. E.; Tseneva, G. Y.; Titov, L. P.; George, R. C.

2002-01-01

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively. PMID:11773096

Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies.

PubMed

Kurohane, Kohta; Nagano, Kyoko; Nakanishi, Katsuhiro; Iwata, Koki; Miyake, Masaki; Imai, Yasuyuki

2014-01-01

Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by size-exclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.

Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

PubMed

Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

2014-05-13

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

PubMed Central

Bayer, A S; Ramos, M D; Menzies, B E; Yeaman, M R; Shen, A J; Cheung, A L

1997-01-01

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis. PMID:9353046

SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

PubMed Central

Wong, Emily S. W.; Hardy, Margaret C.; Wood, David; Bailey, Timothy; King, Glenn F.

2013-01-01

Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the SpiderP website. PMID:23894279

Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

PubMed

Kim, Jung-Beom; Choi, Ok-Kyung; Kwon, Sun-Mok; Cho, Seung-Hak; Park, Byung-Jae; Jin, Na Young; Yu, Yong Man; Oh, Deog-Hwan

2017-08-28

The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua . The hblCDA, nheABC , and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus , which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

PubMed

Liger, D; vanderSpek, J C; Gaillard, C; Cansier, C; Murphy, J R; Leboulch, P; Gillet, D

1997-04-07

We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

PubMed

Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

2015-01-01

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

PubMed

Kirkham, Lea-Ann S; Wiertsema, Selma P; Corscadden, Karli J; Mateus, Tulia; Mullaney, Gemma L; Zhang, Guicheng; Richmond, Peter C; Thornton, Ruth B

2017-03-01

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group ( P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. Copyright © 2017 Kirkham et al.

Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

PubMed

Zhang, Chengxian; Knudsen, David E; Liu, Mei; Robertson, Donald C; Zhang, Weiping

2013-01-01

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains

USDA-ARS?s Scientific Manuscript database

M12X01451, an Enterobacter cloacae strain recently identified from a clinical specimen, produces a new Stx1e that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and information regarding the origin and stability of the stx1e in M12X01451 i...

Increased long-term immunity to Bacillus anthracis protective antigen in mice immunized with a CIA06B-adjuvanted anthrax vaccine.

PubMed

Wui, Seo Ri; Han, Ji Eun; Kim, Yeon Hee; Rhie, Gi-eun; Lee, Na Gyong

2013-04-01

Anthrax is an acute infectious disease caused by Bacillus anthracis. We previously reported that the adjuvant CIA06B, which consists of TLR4 agonist CIA05 and aluminum hydroxide (alum), enhanced the immune response to anthrax protective antigen (PA) in mice. This study was carried out to determine whether CIA06B can enhance long-term immune responses to PA in mice. BALB/c mice were immunized intramuscularly three times at 2-week intervals with recombinant PA alone or PA combined with alum or CIA06B. At 8 and 24 weeks post-immunization, the immunological responses including serum anti-PA IgG antibody titer, toxin-neutralizing antibody titer, splenic cytokine secretion and the frequency of PA-specific memory B cells were assessed. Compared with mice injected with PA alone or PA plus alum, mice injected with PA plus CIA06B had higher titers of serum anti-PA IgG antibodies, and higher frequencies of PA-specific memory B cells and interferon-γ secreting cells. Furthermore, anti-PA antibodies induced by CIA06B were more effective in neutralizing anthrax toxin. These results demonstrated that CIA06B is capable of providing long-term immunity when used as an adjuvant in a PA-based anthrax vaccine.

Immunogenicity and protective efficacy of rotavirus VP8* fused to cholera toxin B subunit in a mouse model.

PubMed

Xue, Miaoge; Yu, Linqi; Jia, Lianzhi; Li, Yijian; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2016-11-01

In attempts to develop recombinant subunit vaccines against rotavirus disease, it was previously shown that the N-terminal truncated VP8* protein, VP8-1 (aa26-231), is a good vaccine candidate when used for immunization in combination with Freund's adjuvant. However, this protein stimulated only weak immune response when aluminum hydroxide was used as an adjuvant. In this study, the nontoxic B subunit of cholera toxin (CTB) was employed as intra-molecular adjuvant to improve the immunogenicity of VP8-1. Both, the N-terminal and C-terminal fusion proteins, were purified to homogeneity, at which stage they formed pentamers, and showed significantly higher immunogenicity and protective efficacy than a VP8-1/aluminum hydroxide mixture in a mouse model. Compared to VP8-1-CTB, CTB-VP8-1 showed higher binding activity to both, GM1 and the conformation sensitive neutralizing monoclonal antibodies specific to VP8. More importantly, CTB-VP8-1 elicited higher titers of neutralizing antibodies and conferred higher protective efficacy than VP8-1-CTB. Therefore, the protein CTB-VP8-1, with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development of an alternative, replication-incompetent, parenterally administered vaccine against rotavirus disease.

Comparing the economic and health benefits of different approaches to diagnosing Clostridium difficile infection.

PubMed

Bartsch, Sarah M; Umscheid, Craig A; Nachamkin, Irving; Hamilton, Keith; Lee, Bruce Y

2015-01-01

Accurate diagnosis of Clostridium difficile infection (CDI) is essential to effectively managing patients and preventing transmission. Despite the availability of several diagnostic tests, the optimal strategy is debatable and their economic values are unknown. We modified our previously existing C. difficile simulation model to determine the economic value of different CDI diagnostic approaches from the hospital perspective. We evaluated four diagnostic methods for a patient suspected of having CDI: 1) toxin A/B enzyme immunoassay, 2) glutamate dehydrogenase (GDH) antigen/toxin AB combined in one test, 3) nucleic acid amplification test (NAAT), and 4) GDH antigen/toxin AB combination test with NAAT confirmation of indeterminate results. Sensitivity analysis varied the proportion of those tested with clinically significant diarrhoea, the probability of CDI, NAAT cost and CDI treatment delay resulting from a false-negative test, length of stay and diagnostic sensitivity and specificity. The GDH/toxin AB plus NAAT approach leads to the timeliest treatment with the fewest unnecessary treatments given, resulted in the best bed management and generated the lowest cost. The NAAT-alone approach also leads to timely treatment. The GDH/toxin AB diagnostic (without NAAT confirmation) approach resulted in a large number of delayed treatments, but results in the fewest secondary colonisations. Results were robust to the sensitivity analysis. Choosing the right diagnostic approach is a matter of cost and test accuracy. GDH/toxin AB plus NAAT diagnosis led to the timeliest treatment and was the least costly. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Serological Studies of Types A, B, and E Botulinal Toxins by Passive Hemagglutination and Bentonite Flocculation

PubMed Central

Johnson, H. M.; Brenner, K.; Angelotti, R.; Hall, H. E.

1966-01-01

Johnson, H. M. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), K. Brenner, R. Angelotti, and H. E. Hall. Serological studies of types A, B, and E botulinal toxins by passive hemagglutination and bentonite flocculation. J. Bacteriol. 91:967–974. 1966.—Formalinized sheep red blood cells (SRBC), sensitized with types A, B, and E botulinal toxoids and toxins by bis-diazotized benzidine (BDB), were tested against A, B, and E antitoxins prepared in horses and rabbits. Type B antitoxin cross-reacted with A toxoid SRBC, but the reciprocal cross-reaction was not observed. E toxin SRBC were specifically agglutinated by E antitoxin. Flocculation of antigen-sensitized bentonite particles was less sensitive in titration of antitoxin than hemagglutination. Also, reciprocal cross-reactions were observed between types A and B antitoxins. Cross-reactions in both serological tests were eliminated by titration of antitoxins in the presence of the heterologous antigens, with no inhibitory effect on the homologous antitoxins. Generally, equine antitoxins were less suitable for agglutinations, especially of antigen-sensitized bentonite particles. Types A, B, and E antitoxins were specifically inhibited by 43, 39, and 245 mouse ld50 of their respective homologous toxins in the hemagglutination-inhibition test. A, B, and E antitoxins were specifically inhibited by 500, 950, and 1,500 mouse ld50 of their respective homologous toxins in bentonite flocculation inhibitions. Formalinized SRBC sensitized with rabbit types A and B antitoxins by BDB were respectively clumped by as little as 0.75 to 1.3 mouse ld50 of A toxin and 2.3 ld50 of B toxin, whereas bentonite particles sensitized by the same antitoxins were specifically clumped by 150 ld50 of A toxin and 630 ld50 of B toxin. E antitoxin sensitization of SRBC or bentonite particles was not successful. Evidence is presented that indicates that the serological procedures are applicable to the detection of botulinal toxins in food. PMID:5326104

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

PubMed

Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C

2015-01-01

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production. Copyright © 2014 Elsevier Ltd. All rights reserved.

The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands.

PubMed

Raj, U; Haq, H; Oshima, Y; Yasumoto, T

1983-01-01

Five species of crabs commonly occurring on Suva barrier reef, Fiji Islands, were tested for the presence of paralytic shellfish toxins. All 35 specimens of Atergatis floridus and all 18 specimens of Zosimus aeneus tested were lethal to mice, whilst none of 12 specimens of Carpilius maculatus, 8 of C. convexus and 10 of Eriphia sebana tested were lethal. A. floridus contained saxitoxin (55--60%), neosaxitoxin (35--40%), gonyautoxin-II (less than 5%) and a new toxin previously found in a toxic dinoflagellate, Pyrodinium bahamense var. compressa, and tentatively coded PBT (1%). Z. aeneus contained the same components, with additional trace amounts of gonyautoxin-I and III, but neosaxitoxin was the major component in this species. Comparison with the results of testing Okinawan specimens of Z. aeneus, A. floridus and Platipodia granulosa suggests that the toxin profile is specific to species.

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection

PubMed Central

Merkel, Tod J; Perera, Pin-Yu; Lee, Gloria M; Verma, Anita; Hiroi, Toyoko; Yokote, Hiroyuki; Waldmann, Thomas A; Perera, Liyanage P

2013-01-01

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response. PMID:23787486

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection.

PubMed

Merkel, Tod J; Perera, Pin-Yu; Lee, Gloria M; Verma, Anita; Hiroi, Toyoko; Yokote, Hiroyuki; Waldmann, Thomas A; Perera, Liyanage P

2013-09-01

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Fatal outbreak of botulism in Greenland.

PubMed

Hammer, Tóra Hedinsdottir; Jespersen, Sanne; Kanstrup, Jakob; Ballegaard, Vibe Cecilie; Kjerulf, Anne; Gelvan, Allan

2015-03-01

Botulism commonly occurs when the anaerobic, gram-positive bacterium Clostridium botulinum, under suitable conditions, produces botulinum neurotoxins. Named A-F, these toxins are the immediate causative agent of the clinical symptoms of symmetrical, descending neurological deficits, including respiratory muscle paralysis. We present five cases of foodborne botulism occurring in Greenland, two with fatal outcome, caused by ingestion of tradionally preserved eider fowl. In the cases of the survivors, antitoxin and supportive care, including mechanical ventilation, were administered. In these cases recovery was complete. Microbiological assays, including toxin neutralization bioassay, demonstrated the presence of neurotoxin E in two survivors. The third survivor was shown by PCR to have the BoNT type E gene in faeces. This is the first report of cases of fatal botulism in Greenland. It underscores the importance of prompt coordinated case management effort in a geographically isolated area such as Greenland.

Glycan Encapsulated Gold Nanoparticles Selectively Inhibit Shiga Toxins 1 and 2

PubMed Central

Kulkarni, Ashish A.; Fuller-Schaefer, Cynthia; Korman, Henry; Weiss, Alison A.; Iyer, Suri S.

2011-01-01

Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae, cause life-threatening conditions that include hemolytic-uremic syndrome (HUS), kidney failure and neurological complications. Cellular entry is mediated by the B subunit of the AB5 toxin, which recognizes cell surface glycolipids present in lipid raft like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These highly soluble glyconanoparticles were nontoxic to the Vero monkey kidney cell line and protected Vero cells from Stx-mediated toxicity in a dose dependent manner. The inhibition is highly dependent on the structure and density of the glycans; selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved. Interestingly, natural variants of Stx2, Stx2c and Stx2d, possessing minimal amino acid variation in the receptor binding site of the B subunit or changes in the A subunit were not neutralized by either the Stx1- or Stx2-specific gold glyconanoparticles. Our results suggest that tailored glyconanoparticles that mimic the natural display of glycans in lipid rafts could serve as potential therapeutics for Stx1 and Stx2. However, a few amino acid changes in emerging Stx2 variants can change receptor specificity, and further research is needed to develop receptor mimics for the emerging variants of Stx2. PMID:20669970

Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana.

PubMed

Garcia, J P; Beingesser, J; Bohorov, O; Bohorova, N; Goodman, C; Kim, D; Pauly, M; Velasco, J; Whaley, K; Zeitlin, L; Roy, C J; Uzal, F A

2014-09-01

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 = 1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 min when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 min post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Evaluation of the ELISA method for cholera toxin determination in Vibrio cholerae cultures].

PubMed

González-Bonilla, C; Gutiérrez-Cogco, L; Moguel-Pech, L; Villanueva-Zamudio, A

1994-01-01

ELISA test was evaluated in 503 cultures of Vibrio cholerae O1 y 303 Non-O1. The cultures were isolated from sewage from different states of México between june 1991 and october 1992. The sensitivity was 100% and specificity was 96%. Only 12 strains of V. cholerae Non-O1 were positive for CT toxin. When these cultures were confirmed by polymerase chain reaction (PCR) for cholera toxin, the results were negative. ELISA test is a good alternative to be used for toxin production in cultures of V. cholerae, it needs confirmation only with O1 negative and Non-O1 positive reactions.

Survey of Genes Encoding Staphylococcal Enterotoxins, Toxic Shock Syndrome Toxin 1, and Exfoliative Toxins in Members of the Staphylococcus sciuri Group

PubMed Central

Dakić, Ivana; Vuković, Dragana; Stepanović, Srdjan; Hauschild, Tomasz; Ježek, Petr; Petráš, Petr; Morrison, Donald

2005-01-01

Genes encoding staphylococcal enterotoxins (sea to see, seg, and seh), toxic shock syndrome toxin 1 (tst), and exfoliative toxins (eta and etb) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely. PMID:16145164

Botulinum toxin therapy in Frey's syndrome: a retrospective study of 440 treatments in 100 patients.

PubMed

Jansen, S; Jerowski, M; Ludwig, L; Fischer-Krall, E; Beutner, D; Grosheva, M

2017-04-01

Frey's syndrome is characterised as sweating, redness and warmth of the parotideal area and is often treated with botulinum toxin A. The objective of this retrospective study was to prove whether the toxin dosage and time-to-treatment intervals change after repeated botulinum toxin injections. The charts of patients, who were treated for Frey's syndrome during the last 16 years, were assessed. Three brands of botulinum toxin A were available for therapy. The Minor test was used to confirm the sweating before each treatment and to determine the toxin dosage. Constant amount of botulinum toxin was injected per cm 2 of the affected area. Patients consulted our department for the next treatment as soon as they felt disturbed by recurring sweating and when the sweating was objectively evident in the Minor test. Time intervals between treatments and injected toxin dosages were assessed. In total, 100 patients received 440 treatments in 16 years. Repeated injections, median 4.0, were carried out in 70.5% of patients. Median time interval to the first injection was 2.8 years. Median time interval between treatments was 12.0 months and showed to be steady (anova, P = .49, F = 1.01). Duration of effect of botulinum toxin on Frey's syndrome was long-lasting and stable with no significantly different time intervals between treatments. The extent of the sweating area did not vary significantly after repeated treatments and required a constant dose of botulinum toxin A. © 2016 John Wiley & Sons Ltd.

A synthetic peptide vaccine directed against the 2ß2-2ß3 loop of domain 2 of protective antigen protects rabbits from inhalation anthrax.

PubMed

Oscherwitz, Jon; Yu, Fen; Cease, Kemp B

2010-09-15

The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.

A high seroprevalence of antibodies to pertussis toxin among Japanese adults: Qualitative and quantitative analyses

PubMed Central

Moriuchi, Takumi; Otsuka, Nao; Hiramatsu, Yukihiro; Shibayama, Keigo

2017-01-01

In 2013, national serosurveillance detected a high seroprevalence of antibodies to pertussis toxin (PT) from Bordetella pertussis among Japanese adults. Thus, we aimed to determine the cause(s) of this high seroprevalence, and analyzed the titers of antibodies to PT and filamentous hemagglutinin (FHA) among adults (35–44 years old), young children (4–7 years old), and older children (10–14 years old). Our quantitative analyses revealed that adults had higher seroprevalences of anti-PT IgG and PT-neutralizing antibodies, and similar titers of anti-FHA IgG, compared to the young and older children. Positive correlations were observed between the titers of PT-neutralizing antibodies and anti-PT IgG in all age groups (rs values of 0.326–0.522), although the correlation tended to decrease with age. The ratio of PT-neutralizing antibodies to anti-PT IgG was significantly different when we compared the serum and purified IgG fractions among adults (p = 0.016), although this result was not observed among young and older children. Thus, it appears that some adults had non-IgG immunoglobulins to PT. Our analyses also revealed that adults had high-avidity anti-PT IgG (avidity index: 63.5%, similar results were observed among the children); however, the adults had lower-avidity anti-FHA IgG (37.9%, p < 0.05). It is possible that low-avidity anti-FHA IgG is related to infection with other respiratory pathogens (e.g., Bordetella parapertussis, Haemophilus influenzae, or Mycoplasma pneumoniae), which produces antibodies to FHA-like proteins. Our observations suggest that these adults had been infected with B. pertussis and other pathogen(s) during their adulthood. PMID:28700751

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins.

PubMed

Torii, Yasushi; Goto, Yoshitaka; Takahashi, Motohide; Ishida, Setsuji; Harakawa, Tetsuhiro; Sakamoto, Takashi; Kaji, Ryuji; Kozaki, Shunji; Ginnaga, Akihiro

2010-01-01

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD(50) test (ip LD(50)). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay. This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD(50), an effect that cannot be detected using the mouse ip LD(50) assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations. Copyright 2009 Elsevier Ltd. All rights reserved.

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

PubMed

Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

2003-02-01

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Non-invasive, epicutaneous immunisation with toxoid in deformable vesicles protects mice against tetanus, chiefly owing to a Th2 response.

PubMed

Chopra, Amla; Cevc, Gregor

2014-06-02

A non-invasive, intra/transcutaneous immunisation of mice with a suitable combination of tetanus toxoid, ultradeformable vesicle (Transfersome®) carrier, and monophosphoryl lipid A adjuvant targets immuno-competent cells in a body and can protect 100% of the tested mice against an otherwise lethal (50×LD50) parenteral tetanus toxin challenge. The late immune response to the epicutaneously applied tetanus toxoid in such vesicles consists chiefly of circulating IgG1 and IgG2b antibody isotypes, indicative of a specific Th2 cellular response bias. Immunisations by subcutaneous injections moreover protect 100% of mice against a similar, otherwise lethal, dose of tetanus toxin. However, the immune response to transcutaneous and invasive immunisation differs. The latter elicits mainly IgG1 and IgG2b as well as IgG2a antibody isotypes, indicative of a mixed Th1/Th2 response. The cytokine response of the intra/transcutaneously and subcutaneously immunised mice reflects the difference in the organ-specific manner. IFN-γ concentration is appreciably increased in the draining lymph nodes and IL-10 in spleen. Since tetanus is a neutral antigen, both the Th1-specific IFN-γ and the Th-2 specific-IL-10 are observable. Copyright © 2014 Elsevier B.V. All rights reserved.

Type C botulism in a commercial turkey farm: a case report.

PubMed

Popp, Christina; Hauck, Rüdiger; Gad, Wael; Hafez, Hafez M

2012-12-01

Botulism is an intoxication caused by exotoxins of Clostridium botulinum. The case of botulism described here occurred on a commercial meat turkey farm with two houses. Toms and hens were maintained in two separate houses, toms in house A and hens in house B. At 10 wk of age, an increase in mortality was observed in the toms located in house A. Clinically the animals presented with paralysis of the legs, wings, and neck. Affected birds were sitting and reluctant to move. Necropsy failed to find any specific lesions. In liver, heart, muscles, crop, and gizzard as well as in intestinal contents, DNA of C. botulinum type C was detected by PCR. The result was confirmed by a mouse lethality neutralization test. During the 2 wk after the onset of the clinical signs the mortality was about 12%. The hens kept in house B did not show any symptoms and remained healthy. Investigations of environmental samples to detect the source of the toxin were not successful. After 2 wk clinical signs and mortality abated. At 16 wk of age, toms again showed the same clinical signs accompanied by raised mortality. Again C. botulinum toxin type C was detected. Within 2 wk the total mortality reached roughly 50%. Based on the "precautionary principle" and in agreement with the local authorities, the birds were euthanatized using CO2 in order to not compromise food safety.

Analyzing titers of antibodies against bacterial and viral antigens, and bacterial toxoids in the intravenous immunoglobulins utilized in Taiwan.

PubMed

Wu, Chi-Yu; Wang, Hsiu-Chi; Wang, Kun-Teng; Yang-Chih Shih, Daniel; Lo, Chi-Fang; Wang, Der-Yuan

2013-03-01

Intravenous immunoglobulin (IVIG) manufactured from human plasma contains IgG as the primary ingredient, and is used for indications such as immunodeficiency syndrome. Available IVIGs in Taiwan are either manufactured from Taiwanese or North American plasma. The effectiveness of the national immunization program of Taiwan can be evaluated by analyzing and comparing IVIG antibody titers that are induced through the corresponding vaccines (tetanus, diphtheria, and pertussis, measles, rubella, hepatitis A, hepatitis B and varicella). Both enzyme-linked immunosorbent assay (ELISA) and the in vitro neutralization test demonstrated that all IVIGs provide adequate clinical protection against diphtheria and tetanus toxins. ELISA results further revealed that plasma of Taiwanese subjects contains higher levels of pertussis toxin and filamentous hemagglutinin antibodies, when compared to foreign IVIGs. This may be related to the later adoption of acellular pertussis vaccine in Taiwan. Antibodies titers against measles, rubella, hepatitis A, and varicella-zoster virus were otherwise low. Low titers of hepatitis B surface antigen antibodies are present in Taiwanese plasma IVIG, indicating immune memory decline or loss. In conclusion, our results show that Taiwanese IVIG contains varying titers of vaccine-induced antibodies, and serves as a guide for future amendments to Taiwan's immunization program. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Extended therapeutic window for post-exposure treatment of ricin intoxication conferred by the use of high-affinity antibodies.

PubMed

Noy-Porat, Tal; Alcalay, Ron; Epstein, Eyal; Sabo, Tamar; Kronman, Chanoch; Mazor, Ohad

2017-03-01

The plant toxin ricin is considered a potential bioterror agent against which there is no available antidote. To date, neutralizing antibodies are the most promising post-exposure treatment for ricin intoxication, yet so far they were shown to be effective only when given within several hours post exposure. As part of an ongoing effort to develop efficient ricin-countermeasures, we tested whether high-affinity antibodies that were previously isolated from immunized non-human primates, may confer effective post-exposure therapy for ricin-intoxicated mice treated at late time-points after exposure. While each antibody is capable of providing high protection rate by itself, a formulation consisting of three neutralizing antibodies that target different epitopes was tested to provide therapeutic coverage against different variants of the malicious pathogen. Indeed, the tri-antibody based cocktail was highly effective, its administration resulting in very high survival rates (>70%) when animals were treated as late as 48 h post exposure and significant protection (>30%) even at 72 h. This study establishes for the first time that anti-ricin antibodies can serve as a highly effective antidote at such late time-points after exposure. From the clinical point of view, the extended therapeutic window documented here is of high importance allowing adequate time to accurately identify the causative agent and may permit initiation of life-saving treatment with these antibodies even after the onset of clinical signs. Copyright © 2017 Elsevier Ltd. All rights reserved.

PROTECTION OF MICE AGAINST IRRADIATION AND TETANUS BY HOMOLOGOUS BONE MARROW CELLS FROM HYPERIMMUNIZED DONORS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoloff, I.L.; Weiss, A.J.

1963-07-01

Female mice of inbred strains (101 x C3H, BDF, C57B1, Balb/C, C3H, CBA, and LAF) were immunized with 0.2 ml of alum-precipitated tetanus toxoid subcutaneously, followed in 3 weeks by 0.2 ml of fluid toxoid intravenously. Four days after the last injection the marrow was mechanically dispersed and 10- 20 million marrow cells were inoculated intravenously into mice that had received on the previous day a lethal dose of whole-body x irradiation. The LD/sub 96/ for 30 days of each host strain was: BDF, 950 r; LAF, 950 r; 101 x C3H, 900 r; Balb/C, 800 r; C3H, 800 r;more » C57B1, 800 r; and CBA, 700 r. Mice in which isologous bone marrow cells from hyperimmunized donors were transferred to irradiated hosts showed a high degree of protection against irradiation in all strains studied. The percentage of 30-day irradiation survivors follows: C3H, 100%; 101 x C3H, 100%; CBA, 90%; BDF, 90%; Balb/C, 60%; and C57B1, 70%. There were no survivors among groups irradiated but not protected with bone marrow. The percentage of 7- day survivors after toxin challenge for each of 4 different strains receiving isologous cells from hyperimmunized donors ranged between 87 and 100%. Normal mice, similar in weight to the experimental groups (called toxin controls) all died of tetanus within 48 hr of challenge with toxin. Other results showed that homologous disease does not interfere significantly with the in vivo neutralization of tetanus toxin by antitoxin. It was concluded that homologous disease is a clinical entity which, in some donor-host combinations, is associated with a host-vs-graft reaction and, in one strain combination so far tested, is associated with a graft-vshost reaction. The experiments showed that the genetic relation between donor and host is a factor in determining which type of immunologic reaction may occur. (TCO)« less

CONDUCT AN INVESTIGATION OF CIGUATERA POISON

DTIC Science & Technology

Research on ciguatera toxin has resulted in the development of an efficient method for extracting the toxin from the musculature of fish. Procedures...solution and adsorption of ciguatera toxin on silicic ac d. More precise fractionation of ciguatera toxin is accomplished on a silicic acid column...developed in stepwise fashion by ether containing small percentages of methanol.As a method for fractionation and as the basis for a test for ciguatera

Collaborative Research Program on Seafood Toxins

DTIC Science & Technology

1988-08-14

Crystallographic Structures of Saxitoxins Cl and C2 Appendix C: Collaborative Research Program an Seafcod Toxins Progress Report on Ciguatera and Related...radioimmunoassay for PSP were also evalumted. The Hokama stick test for ciguatera toxin was also evaluated. 4. initiate Studies on the Accumulation...tco•d which caie a form of b-mnn poisoning referred to as ciguatera . The respcnsible toxins originate from ll1ular rine algae of the division

Dinoflagellate Toxins Responsible for Ciguatera Food Poisoning

DTIC Science & Technology

1987-12-10

the occurrence and importance of secondary toxins (12, 13, 14, 15, 16 and 17). Hutner and McLaughlin (18) suggested that the zooxanthellae ...argued that zooxanthellae were unlikely prospects because very few fishes eat coral and saxitoxin (STX = a paralytic shellfish poison which originates...of the toxins was not based on tests of zooxanthellae species but rather on the assumption that all dinoflagellate toxins would be expected to be

Comparison of T-2 Toxin and HT-2 Toxin Distributed in the Skeletal System with That in Other Tissues of Rats by Acute Toxicity Test.

PubMed

Yu, Fang Fang; Lin, Xia Lu; Yang, Lei; Liu, Huan; Wang, Xi; Fang, Hua; Lammi, ZMikko J; Guo, Xiong

2017-11-01

Twelve healthy rats were divided into the T-2 toxin group receiving gavage of 1 mg/kg T-2 toxin and the control group receiving gavage of normal saline. Total relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone, knee joints, and costal cartilage) were significantly higher than those in the heart, liver, and kidneys (P < 0.05). The relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone and costal cartilage) were also significantly higher than those in the heart, liver, and kidneys. The rats administered T-2 toxin showed rapid metabolism compared with that in rats administered HT-2 toxin, and the metabolic conversion rates in the different tissues were 68.20%-90.70%. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

PubMed Central

Avril, Arnaud; Miethe, Sebastian; Derman, Yagmur; Selby, Katja; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Korkeala, Hannu; Sesardic, Dorothea; Popoff, Michel R.

2017-01-01

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans. PMID:28974033

Comparison of non-O157 Shiga toxin-producing E. coli detection systems

USDA-ARS?s Scientific Manuscript database

Category: methodology improvements Objective: To identify strengths and weaknesses of commercial Shiga toxin-producing E. coli detection systems and kits in a side by side fashion. Experimental Design: Three commercial Shiga toxin-producing E. coli detection tests (BAX, GDS, and GeneDisc) and two t...

Thermal stability and structural changes in bacterial toxins responsible for food poisoning

PubMed Central

Regenthal, Paulina; Hansen, Jesper S.; André, Ingemar

2017-01-01

The staphylococcal enterotoxins (SEs) are secreted by the bacteria Staphylococcus aureus and are the most common causative agent in staphylococcal food poisoning. The staphylococcal enterotoxin A (SEA) has been associated with large staphylococcal food poisoning outbreaks, but newer identified SEs, like staphylococcal enterotoxin H (SEH) has recently been shown to be present at similar levels as SEA in food poisoning outbreaks. Thus, we set out to investigate the thermo-stability of the three-dimensional structures of SEA, SEH and staphylococcal enterotoxin E (SEE), since heat inactivation is a common method to inactivate toxins during food processing. Interestingly, the investigated toxins behaved distinctly different upon heating. SEA and SEE were more stable at slightly acidic pH values, while SEH adopted an extremely stable structure at neutral pH, with almost no effects on secondary structural elements upon heating to 95°C, and with reversible formation of tertiary structure upon subsequent cooling to room temperature. Taken together, the data suggests that the family of staphylococcal enterotoxins have different ability to withstand heat, and thus the exact profile of heat inactivation for all SEs causing food poisoning needs to be considered to improve food safety. PMID:28207867

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile

PubMed Central

Hong, Huynh A.; Hitri, Krisztina; Hosseini, Siamand; Kotowicz, Natalia; Bryan, Donna; Mawas, Fatme; Wilkinson, Anthony J.; van Broekhoven, Annie; Kearsey, Jonathan

2017-01-01

ABSTRACT Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26–39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26–39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26–39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile. These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26–39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI. PMID:28167669

Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli.

PubMed

Tsukuda, Miyuki; Nakashima, Nobutaka; Miyazaki, Kentaro

2015-11-01

Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited. Here we developed a novel counterselection method in E. coli based on antisense RNA (asRNA) technology directed against toxin-antitoxin (TA) modules. Under normal conditions, excess antitoxin neutralizes its cognate toxin and thus the module is stably maintained in the genome. We hypothesised that repression of an antitoxin gene would perturb cell growth due to the toxin being released. We designed asRNAs corresponding to all 19 type II antitoxins encoded in the E. coli genome. asRNAs were then conditionally expressed; repression of MqsA in the MqsR/MqsA module had the greatest inhibitory effect, followed by RnlB in the RnlA/RnlB module. The utility of asRNA(MqsA) as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria.

PubMed

Engler, K H; Efstratiou, A

2000-04-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.

Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria

PubMed Central

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day. PMID:10747112

Evaluation of coproexamination as a diagnostic test for avian botulism

USGS Publications Warehouse

Jensen, Wayne I.

1981-01-01

Fecal extracts and blood sera from 113 ducks showing clinical signs of botulism were examined for Clostridium botulinum type C toxin by means of the mouse toxicity test to evaluate coproexamination as a diagnostic procedure, as compared with demonstration of toxin in serum. When death of test mice unprotected with type specific antitoxin (while protected controls survived) was the criterion, 78.8% of the sera and 5.3% of the fecal extracts were positive. When characteristic signs of intoxication in the unprotected mice was included as evidence of toxin in the specimens, these percentages increased to 86.7 and 6.2, respectively.Fecal specimens were collected hourly for the first 6 h after peroral dosing of eight mallards (Anas platyrhynchos) with 1.0 LD50, of type C toxin and at 24, 48, and 72 h from birds surviving that long. From 2 to 4 toxin-positive specimens were passed by all eight ducks during the first 6 h, five specimens were positive at 24 h, and three were positive at 48 h. Only three specimens were collected at 72 h, all of which were negative. These findings suggest that attempts to detect toxin in the feces of wild ducks might have been more successful had the birds been captured earlier in the course of the disease.

Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria

PubMed Central

Efstratiou, Androulla; Engler, Kathryn H.; Dawes, Charlotte S.; Sesardic, Dorothea

1998-01-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended. PMID:9774560

Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria.

PubMed

Efstratiou, A; Engler, K H; Dawes, C S; Sesardic, D

1998-11-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.

Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

DTIC Science & Technology

2013-10-01

acids. These sites constitute a variable environment, with the effect of mutations largely isolated to effects on interactions with the P4 side chain. 2...desires to cut. We observe, however, sequence-specific cleavage is much more subtle, depending upon how side chain interactions influence not only...first five substrate amino acids on the acyl side of the scissile bond (denoted P1 through P5, numbering from the scissile bond toward the N-terminus

Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

DTIC Science & Technology

2012-10-14

effect of mutations largely isolated to effects on interactions with the P4 side chain. 2) Most mutations at some sites (e.g. 126, 128) decrease...to cut. We observe, however, sequence-specific cleavage is much more subtle, depending upon how side chain interactions influence not only ground...five substrate amino acids on the acyl side of the scissile bond (denoted P1 through P5, numbering from the scissile bond toward the N-terminus of the

Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its Gene

DTIC Science & Technology

1987-03-01

other venoms and examine their toxin neutral- izing ability. The amino acid sequences of both crotoxin subunits were determined Is a prelude to cloning...be examined for their potential as anti-idiotype vaccines The complete amino acid sequence of the basic subunit and two of the three dic subunit chains...of crotoxin from the venom of C.d. terrificus has been de rmined. Sequence comparison data suggest that the non-toxic, acidic subunit was derived

Snake venomics of Micrurus alleni and Micrurus mosquitensis from the Caribbean region of Costa Rica reveals two divergent compositional patterns in New World elapids.

PubMed

Fernández, Julián; Vargas-Vargas, Nancy; Pla, Davinia; Sasa, Mahmood; Rey-Suárez, Paola; Sanz, Libia; Gutiérrez, José María; Calvete, Juan J; Lomonte, Bruno

2015-12-01

Protein composition, toxicity, and neutralization of the venoms of Micrurus alleni and Micrurus mosquitensis, two sympatric monadal coral snakes found in humid environments of the Caribbean region of Costa Rica, were studied. Proteomic profiling revealed that these venoms display highly divergent compositions: the former dominated by three-finger toxins (3FTx) and the latter by phospholipases A2 (PLA2). Protein family abundances correlated with enzymatic and toxic characteristics of the venoms. Selective inhibition experiments showed that PLA2s play only a marginal role in the lethal effect of M. alleni venom, but have a major role in M. mosquitensis venom. Proteomic data gathered from other Micrurus species evidenced that the two divergent venom phenotypes are recurrent, and may constitute a general trend across New World elapids. Further, M. mosquitensis, but not M. alleni, venom contains PLA2-like/Kunitz-type inhibitor complex(es) that resemble the ASIC1a/2-activating MitTx heterodimeric toxin isolated from Micrurus tener venom. The evolutionary origin and adaptive relevance of the puzzling phenotypic variability of Micrurus venoms remain to be understood. An antivenom against the PLA2-predominant Micrurus nigrocinctus venom strongly cross-recognized and neutralized M. mosquitensis venom, but only weakly M. alleni venom. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sub-Lethal Dose of Shiga Toxin 2 from Enterohemorrhagic Escherichia coli Affects Balance and Cerebellar Cytoarchitecture

PubMed Central

Pinto, Alipio; Cangelosi, Adriana; Geoghegan, Patricia A.; Tironi-Farinati, Carla; Brener, Gabriela J.; Goldstein, Jorge

2016-01-01

Shiga toxin producing Escherichia coli may damage the central nervous system before or concomitantly to manifested hemolytic–uremic syndrome symptoms. The cerebellum is frequently damaged during this syndrome, however, the deleterious effects of Shiga toxin 2 has never been integrally reported by ultrastructural, physiological and behavioral means. The aim of this study was to determine the cerebellar compromise after intravenous administration of a sub-lethal dose of Shiga toxin 2 by measuring the cerebellar blood–brain barrier permeability, behavioral task of cerebellar functionality (inclined plane test), and ultrastructural analysis (transmission electron microscope). Intravenous administration of vehicle (control group), sub-lethal dose of 0.5 and 1 ηg of Stx2 per mouse were tested for behavioral and ultrastructural studies. A set of three independent experiments were performed for each study (n = 6). Blood–brain barrier resulted damaged and consequently its permeability was significantly increased. Lower scores obtained in the inclined plane task denoted poor cerebellar functionality in comparison to their controls. The most significant lower score was obtained after 5 days of 1 ηg of toxin administration. Transmission electron microscope micrographs from the Stx2-treated groups showed neurons with a progressive neurodegenerative condition in a dose dependent manner. As sub-lethal intravenous Shiga toxin 2 altered the blood brain barrier permeability in the cerebellum the toxin penetrated the cerebellar parenchyma and produced cell damaged with significant functional implications in the test balance. PMID:26904009

Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus from Retail Raw Chicken Meat.

PubMed

Li, Suixia; Wang, Panpan; Zhao, Jialin; Zhou, Luhong; Zhang, Pengfei; Fu, Chengyu; Meng, Jianghong; Wang, Xin

2018-04-01

The aim of this study was to investigate the toxin gene profile and antimicrobial resistance of Staphylococcus aureus isolates from raw chicken in the People's Republic of China. In total, 289 S. aureus isolates were characterized by antimicrobial susceptibility testing, and genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin were revealed by PCR. Overall, 46.0% of the isolates were positive for one or more toxin genes. A high proportion of toxin genes were pvl (26.6%), followed by sej (12.5%), sea (9.0%), seh (8.3%), seb (6.9%), sec (6.9%), sed (4.8%), sei (3.1%), and see (2.4%). None of the isolates harbored seg, tsst-1, or exfoliative toxin genes. In total, 29 toxin gene profiles were obtained, and pvl (10.7%) was the most frequent genotype, followed by sea (5.9%), seb (4.8%), and sej (4.2%). Furthermore, 99.7% of the strains were resistant to at least one of the tested antimicrobial agents, and 87.2% of them displayed multidrug resistance. Resistance was most frequently observed to trimethoprim-sulfamethoxazole and erythromycin (86.2% for each), followed by tetracycline (69.9%), amoxicillin-clavulanic acid (45.0%), and ampicillin (42.6%). None of the strains were resistant to vancomycin. This study indicates that S. aureus isolates from raw chicken harbored multiple toxin genes and exhibited multiple antimicrobial resistance, which represents a potential health hazard for consumers.

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

PubMed

Tkaczyk, C; Kasturirangan, S; Minola, A; Jones-Nelson, O; Gunter, V; Shi, Y Y; Rosenthal, K; Aleti, V; Semenova, E; Warrener, P; Tabor, D; Stover, C K; Corti, D; Rainey, G; Sellman, B R

2017-08-01

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb. Copyright © 2017 Tkaczyk et al.

Physical and chemical processes promoting dominance of the toxic cyanobacterium Cylindrospermopsis raciborskii

NASA Astrophysics Data System (ADS)

Burford, Michele A.; Davis, Timothy W.

2011-07-01

The freshwater cyanobacterium, Cylindrospermopsis raciborskii (Wo'oszyńska) Seenayya and Subba Raju is a common species in lakes and reservoirs globally. In some areas of the world it can produce cyto- and hepatotoxins (cylindrospermopsins, saxitoxins), making blooms of this species a serious health concern for humans. In the last 10-15 years, there has been a considerable body of research conducted on the ecology, physiology and toxin production of this species and this paper reviews these studies with a focus on the cylindrospermopsin (CYN)-producing strains. C. raciborskii has low light requirements, close to neutral buoyancy, and a wide temperature tolerance, giving it the capacity to grow in many lentic waterbodies. It also has a flexible strategy with respect to nitrogen (N) utilisation; being able to switch between utilising fixed and atmospheric N as sources of N fluctuate. Additionally this species has a high phosphate (DIP) affinity and storage capacity. Like many cyanobacteria, it also has the capacity to use dissolved organic phosphorus (DOP). Changes in nutrient concentrations, light levels and temperature have also been found to affect production of the toxin CYN by this species. However, optimal toxin production does not necessarily occur when growth rates are optimal. Additionally, different strains of C. raciborskii vary in their cell quota of CYN, making it difficult to predict toxin concentrations, based on C. raciborskii cell densities. In summary, the ecological flexibility of this organism means that controlling blooms of C. raciborskii is a difficult undertaking. However, improved understanding of factors promoting the species and toxin production by genetically capable strains will lead to improved predictive models of blooms.

Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

PubMed

Dong, Na; Luo, Longlong; Wu, Junhua; Jia, Peiyuan; Li, Qian; Wang, Yuxia; Gao, Zhongcai; Peng, Hui; Lv, Ming; Huang, Chunqian; Feng, Jiannan; Li, Hua; Shan, Junjie; Han, Gang; Shen, Beifen

2015-07-31

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20μg/kg) even at 6h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

PubMed Central

Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

2015-01-01

Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples

USDA-ARS?s Scientific Manuscript database

Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their effectiveness in detecting and differentiating between Shiga toxin 1 and 2 (Stx1 and Stx2) produced by Shiga toxin-producing E. coli (STEC) inoculated into food and water samples. Each kit incorporated monoclonal antibodies ...

Clinical recovery and circulating botulinum toxin type F in adult patient.

PubMed

Sobel, Jeremy; Dill, Tracy; Kirkpatrick, Christina L; Riek, Laurel; Luedtke, Patrick; Damrow, Todd A

2009-06-01

A 56-year-old woman in Helena, Montana, USA, who showed clinical signs of paralysis, received antitoxins to botulinum toxins A, B, and E within 24 hours; nevertheless, symptoms progressed to complete quadriplegia. On day 8, she began moving spontaneously, even though blood tests later showed botulinum toxin type F remained.

9 CFR 121.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Exemptions for overlap select agents and toxins. 121.6 Section 121.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... select agent or toxin that is contained in a specimen presented for proficiency testing will be exempt...

9 CFR 121.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Exemptions for overlap select agents and toxins. 121.6 Section 121.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... select agent or toxin that is contained in a specimen presented for proficiency testing will be exempt...

9 CFR 121.5 - Exemptions for VS select agents and toxins.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Exemptions for VS select agents and toxins. 121.5 Section 121.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... toxin that is contained in a specimen presented for proficiency testing will be exempt from the...

9 CFR 121.5 - Exemptions for VS select agents and toxins.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Exemptions for VS select agents and toxins. 121.5 Section 121.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... toxin that is contained in a specimen presented for proficiency testing will be exempt from the...

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

PubMed

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-04-21

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus

PubMed Central

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-01-01

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses. PMID:27110810

Characterization of the Deep-Sea Streptomyces sp. SCSIO 02999 Derived VapC/VapB Toxin-Antitoxin System in Escherichia coli.

PubMed

Guo, Yunxue; Yao, Jianyun; Sun, Chenglong; Wen, Zhongling; Wang, Xiaoxue

2016-07-01

Toxin-antitoxin (TA) systems are small genetic elements that are ubiquitous in prokaryotes. Most studies on TA systems have focused on commensal and pathogenic bacteria; yet very few studies have focused on TAs in marine bacteria, especially those isolated from a deep sea environment. Here, we characterized a type II VapC/VapB TA system from the deep-sea derived Streptomyces sp. SCSIO 02999. The VapC (virulence-associated protein) protein belongs to the PIN (PilT N-terminal) superfamily. Overproduction of VapC strongly inhibited cell growth and resulted in a bleb-containing morphology in E. coli. The toxicity of VapC was neutralized through direct protein-protein interaction by a small protein antitoxin VapB encoded by a neighboring gene. Antitoxin VapB alone or the VapB/VapC complex negatively regulated the vapBC promoter activity. We further revealed that three conserved Asp residues in the PIN domain were essential for the toxic effect of VapC. Additionally, the VapC/VapB TA system stabilized plasmid in E. coli. Furthermore, VapC cross-activated transcription of several TA operons via a partially Lon-dependent mechanism in E. coli, and the activated toxins accumulated more preferentially than their antitoxin partners. Collectively, we identified and characterized a new deep sea TA system in the deep sea Streptomyces sp. and demonstrated that the VapC toxin in this system can cross-activate TA operons in E. coli.

Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah).

PubMed

Tan, Choo Hock; Tan, Kae Yi; Fung, Shin Yee; Tan, Nget Hong

2015-09-10

The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS. Transcriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A2 (PLA2s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). Seventeen toxin families exhibited low mRNA expression, including hyaluronidase, DPP-IV and 5'-nucleotidase that were not previously reported in the venom-gland transcriptome of a Balinese O. hannah. On the other hand, the MOh proteome includes 3FTxs, the most abundantly expressed proteins in the venom (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 non-conventional 3FTx. Neurotoxins comprise the major 3FTxs in the MOh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. The presence of toxic enzymes such as LAAOs, SVMPs and PLA2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) are in agreement with the poor cross-neutralization activity of N. kaouthia antivenom used against MOh venom. Besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5'-nucleotidase, and DPP-IV in the venom proteome suggests its probable hypotensive action in subduing prey. This study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). The results correlate with the pathophysiological actions of MOh venom, and dispute the use of Naja cobra antivenoms to treat MOh envenomation. The findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom.

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

PubMed Central

Ruan, Xiaosai; Sack, David A.; Zhang, Weiping

2015-01-01

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development. PMID:25803825

Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

PubMed

Ruan, Xiaosai; Sack, David A; Zhang, Weiping

2015-01-01

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

Role of Botulinum Toxin in Depression.

PubMed

Parsaik, Ajay K; Mascarenhas, Sonia S; Hashmi, Aqeel; Prokop, Larry J; John, Vineeth; Okusaga, Olaoluwa; Singh, Balwinder

2016-03-01

The goal of this review was to consolidate the evidence concerning the efficacy of botulinum toxin type A (onabotulinumtoxinA) in depression. We searched MEDLINE, EMBASE, Cochrane, and Scopus through May 5, 2014, for studies evaluating the efficacy of botulinum toxin A in depression. Only randomized controlled trials were included in the meta-analysis. A pooled mean difference in primary depression score, and pooled odds ratio for response and remission rate with 95% confidence interval (CI) were estimated using the random-effects model. Heterogeneity was assessed using Cochran Q test and χ statistic. Of the 639 articles that were initially retrieved, 5 studies enrolling 194 subjects (age 49±9.6 y) were included in the systematic review, and 3 randomized controlled trials enrolling 134 subjects were included in the meta-analysis. The meta-analysis showed a significant decrease in mean primary depression scores among patients who received botulinum toxin A compared with placebo (-9.80; 95% CI, -12.90 to -6.69) with modest heterogeneity between the studies (Cochran Q test, χ=70). Response and remission rates were 8.3 and 4.6 times higher, respectively, among patients receiving botulinum toxin A compared with placebo, with no heterogeneity between the studies. The 2 studies excluded from the meta-analysis also found a significant decrease in primary depression scores in patients after receiving botulinum toxin A. A few subjects had minor side effects, which were similar between the groups receiving botulinum toxin and those receiving placebo. This study suggests that botulinum toxin A can produce significant improvement in depressive symptoms and is a safe adjunctive treatment for patients receiving pharmacotherapy for depression. Future trials are needed to evaluate the antidepressant effect per se of botulinum toxin A and to further elucidate the underlying antidepressant mechanism of botulinum toxin A.

77 FR 9888 - Shiga Toxin-Producing Escherichia coli

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-21

... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... routine verification sampling and testing for raw beef manufacturing trimmings for six non-O157 Shiga... announced in September 2011 plans to test certain raw beef products for these six STEC serogroups in...

Mucosal and systemic adjuvant effects of cholera toxin and Cry1Ac protoxin on the specific antibody response to HIV-1 C4/V3 peptides are different and depend on the antigen co-administered.

PubMed

Esquivel-Pérez, Rita; Moreno-Fierros, Leticia

2005-01-01

Evidence from several sources support the assertion that cholera toxin (CT) is a potent immunogen and mucosal adjuvant; however there are also reports showing its lack of adjuvanticity against some antigens. Cry1Ac protoxin also exerts adjuvant effects in the antibody responses to proteins and polysaccharides but its adjuvanticity with regard to peptide vaccines had not been tested. To probe whether the adjuvant effects of these proteins varied depending on the antigen co-administered, we evaluated antipeptide antibody responses in serum and mucosal samples (vaginal, intestinal, and pulmonary) of mice that were immunized by intranasal or intraperitoneal routes with one of two distinct hybrid C4/V3 HIV peptides, either alone or with CT or Cry1Ac. The tested HIV 1 peptides differed in two aminoacids, T1SP10MN(A) was modified at the SP10 region by the substitution of the isoleucines 12 and 14 for cysteines and was called modified (m)T1SP10MN(A). Our data indicate that the adjuvant effects of CT and Cry1Ac are different. In addition they vary depending on the antigen co-administered and the number of antigen doses, because after three doses moderate adjuvant effects of CT and Cry1Ac on anti-peptide serum and mucosal antibody responses were observed only against the mT1SP10MN(A). In contrast, to attain significant adjuvant effects against the T1SP10MN, four doses were required. Interestingly we found that modification of the HIV peptide increases its immunogenicity without altering the principal neutralizing determinant (SP10).

Thermodynamics of membrane insertion and refolding of the diphtheria toxin T-domain

PubMed Central

Vargas-Uribe, Mauricio; Rodnin, Mykola V.; Öjemalm, Karin; Holgado, Aurora; Kyrychenko, Alexander; Nilsson, IngMarie; Posokhov, Yevgen O.; Makhatadze, George; von Heijne, Gunnar; Ladokhin, Alexey S.

2014-01-01

The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3–5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately −8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between −9.5 to −12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain. PMID:25281329

FOLDING OF DIPHTHERIA TOXIN T-DOMAIN IN THE PRESENCE OF AMPHIPOLS AND FLUORINATED SURFACTANTS: TOWARD THERMODYNAMIC MEASUREMENTS OF MEMBRANE PROTEIN FOLDING

PubMed Central

Kyrychenko, Alexander; Rodnin, Mykola V.; Vargas-Uribe, Mauricio; Sharma, Shivaji K.; Durand, Grégory; Pucci, Bernard; Popot, Jean-Luc; Ladokhin, Alexey S.

2011-01-01

Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which tend to destabilize them, however. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homo-polymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of vesicle content leakage indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. PMID:21945883

Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

DTIC Science & Technology

2014-06-01

protein and producing adequate amounts for in vitro testing. 15. SUBJECT TERMS- Botulinum toxin , ubiquitin, chimeric toxin light chains, LcA, LcE...that confer stability to LCs of botulinum toxin can be assessed by mutation of dileucine residues and systematic deletion of residues from LcA-LcE...cells. So What? Currently, there is no cure for botulinum poisoning once the toxin has entered a neuron. Moreover, the half-life of BoNT/A is very

ALTERATIONS OF BACTERIAL TOXINS BY THE CONSTITUENTS OF CHAMOMILE AND HORSERADISH. VI. STUDY ON THE QUESTION OF THE CONVERSION OF LETHALLY ACTING STAPHYLOCOCCI TOXIN INTO TOXOID UNDER THE ACTION OF CHAMOMILES AND HORSERADISH CONSTITUENS

DTIC Science & Technology

On the basis of the investigations it is concluded that the chamomile and horseradish constituents tested, in addition to their hitherto known effects, very probably also have a detoxicating effect upon bacteria toxins.

Attenuation of Ricin Toxin under Ambient Conditions and ...

EPA Pesticide Factsheets

Report This study focused on the attenuation of ricin toxin on six types of materials representative of a mail sorting facility and/or indoor building materials. Attenuation tests were conducted under various combinations of temperature, relative humidity (RH), and contact time, using two forms of ricin toxin: a commercially-available pure preparation and a crude preparation from castor beans.

Clinical Recovery and Circulating Botulinum Toxin Type F in Adult Patient

PubMed Central

Dill, Tracy; Kirkpatrick, Christina L.; Riek, Laurel; Luedtke, Patrick; Damrow, Todd A.

2009-01-01

A 56-year-old woman in Helena, Montana, USA, who showed clinical signs of paralysis, received antitoxins to botulinum toxins A, B, and E within 24 hours; nevertheless, symptoms progressed to complete quadriplegia. On day 8, she began moving spontaneously, even though blood tests later showed botulinum toxin type F remained. PMID:19523306

9 CFR 121.5 - Exemptions for VS select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Exemptions for VS select agents and toxins. 121.5 Section 121.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... agent or toxin that is contained in a specimen presented for proficiency testing will be exempt from the...

9 CFR 121.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Exemptions for overlap select agents and toxins. 121.6 Section 121.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... presented for proficiency testing will be exempt from the requirements of this part for such agent or toxin...

9 CFR 121.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Exemptions for overlap select agents and toxins. 121.6 Section 121.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... presented for proficiency testing will be exempt from the requirements of this part for such agent or toxin...

9 CFR 121.5 - Exemptions for VS select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Exemptions for VS select agents and toxins. 121.5 Section 121.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... agent or toxin that is contained in a specimen presented for proficiency testing will be exempt from the...

9 CFR 121.5 - Exemptions for VS select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Exemptions for VS select agents and toxins. 121.5 Section 121.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... agent or toxin that is contained in a specimen presented for proficiency testing will be exempt from the...

9 CFR 121.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Exemptions for overlap select agents and toxins. 121.6 Section 121.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... presented for proficiency testing will be exempt from the requirements of this part for such agent or toxin...

Use of monoclonal antibodies as an effective strategy for treatment of ciguatera poisoning.

PubMed

Inoue, Masayuki; Lee, Nayoung; Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

2009-06-01

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

Protective Antigen (PA) and Toxin Neutralization (TNA) Antibody Patterns in Anthrax Vaccinees Undergoing Serial Plasmapheresis

DTIC Science & Technology

2005-03-28

regulatory agencies to ensure the well- being of donors. Studies have shown that weekly plasma FIG. 1. IgG antibody to B . anthracis PA concentrations in...plasmapheresis (P 0.0007) ( B ). 716 PITTMAN ET AL. CLIN. DIAGN. LAB. IMMUNOL. donors may experience declines in total IgG , IgA, and IgM... B ). VOL. 12, 2005 ANTHRAX ANTIBODIES IN IMMUNE PLASMA 717 FIG. 3. Comparative rates of decline between IgG antibody to B . anthracis PA

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle.

PubMed

Gregory, A R; Ellis, T M; Jubb, T F; Nickels, R J; Cousins, D V

1996-02-01

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.

Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.

PubMed

De Zoysa, Aruni; Efstratiou, Androulla; Mann, Ginder; Harrison, Timothy G; Fry, Norman K

2016-12-01

Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.

Immunization studies with attenuated strains of Bacillus anthracis.

PubMed Central

Ivins, B E; Ezzell, J W; Jemski, J; Hedlund, K W; Ristroph, J D; Leppla, S H

1986-01-01

Live, attenuated strains of Bacillus anthracis lacking either the capsule plasmid pXO2, the toxin plasmid pXO1, or both were tested for their efficacy as vaccines against intravenous challenge with anthrax toxin in Fischer 344 rats and against aerosol or intramuscular challenge with virulent anthrax spores in Hartley guinea pigs. Animals immunized with toxigenic, nonencapsulated (pXO1+, pXO2-) strains survived toxin and spore challenge and demonstrated postimmunization antibody titers to the three components of anthrax toxin (protective antigen, lethal factor, and edema factor). Immunization with two nontoxigenic, encapsulated (pXO1-, pXO2+), Pasteur vaccine strains neither provided protection nor elicited titers to any of the toxin components. Therefore, to immunize successfully against anthrax toxin or spore challenge, attenuated, live strains of B. anthracis must produce the toxin components specified by the pXO1 plasmid. PMID:3084383

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions.

PubMed

Koehler, Susanne M; Buyuk, Fatih; Celebi, Ozgur; Demiraslan, Hayati; Doganay, Mehmet; Sahin, Mitat; Moehring, Jens; Ndumnego, Okechukwu C; Otlu, Salih; van Heerden, Henriette; Beyer, Wolfgang

2017-07-12

Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody.

PubMed

Hoshino, Yu; Koide, Hiroyuki; Urakami, Takeo; Kanazawa, Hiroaki; Kodama, Takashi; Oku, Naoto; Shea, Kenneth J

2010-05-19

We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

Evaluation of Passive Samplers as a Monitoring Tool for Early Warning of Dinophysis Toxins in Shellfish

PubMed Central

Pizarro, Gemita; Moroño, Ángeles; Paz, Beatriz; Franco, José M.; Pazos, Yolanda; Reguera, Beatriz

2013-01-01

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography–mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week−1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks. PMID:24152559

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Cyanotoxins from Black Band Disease of Corals and from Other Coral Reef Environments

PubMed Central

Sekar, Raju; Richardson, Laurie L.

2012-01-01

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease. PMID:19554362

Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth.

PubMed

Weese, J S; Cote, N M; deGannes, R V G

2003-11-01

Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

PubMed

Sesardic, D; Prior, C; Daas, A; Buchheit, K H

2003-07-01

A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

Cyanotoxins from black band disease of corals and from other coral reef environments.

PubMed

Gantar, Miroslav; Sekar, Raju; Richardson, Laurie L

2009-11-01

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

PubMed Central

Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong

2016-01-01

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents. PMID:27058956

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia.

PubMed

Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong

2016-04-01

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a 'pan-specific' AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a 'pan-specific' AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund's adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.

Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model.

PubMed

Amog, P U; Manjuprasanna, V N; Yariswamy, M; Nanjaraj Urs, A N; Joshi, Vikram; Suvilesh, K N; Nataraju, A; Vishwanath, Bannikuppe Sannanaik; Gowda, T V

2016-11-01

Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A 2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC 50  =   36.32 μg, p < 0.0001) and hyaluronidase (IC 50  =   91.95 μg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED 50  =   26.37 μg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED 50  =   37.5 μg, p < 0.0001) and lactate dehydrogenase (ED 50  =   31.44 μg, p = 0.0021) levels at 1:50 w/w. ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.

Successful refolding and NMR structure of rMagi3: A disulfide-rich insecticidal spider toxin.

PubMed

Titaux-Delgado, Gustavo; Carrillo, Elisa; Mendoza, Angeles; Mayorga-Flores, Marlen; Escobedo-González, Fátima C; Cano-Sánchez, Patricia; López-Vera, Estuardo; Corzo, Gerardo; Del Rio-Portilla, Federico

2018-03-01

The need for molecules with high specificity against noxious insects leads the search towards spider venoms that have evolved highly selective toxins for insect preys. In this respect, spiders as a highly diversified group of almost exclusive insect predators appear to possess infinite potential for the discovery of novel insect-selective toxins. In 2003, a group of toxins was isolated from the spider Macrothele gigas and the amino acid sequence was reported. We obtained, by molecular biology techniques in a heterologous system, one of these toxins. Purification process was optimized by chromatographic methods to determine the three-dimensional structure by nuclear magnetic resonance in solution, and, finally, their biological activity was tested. rMagi3 resulted to be a specific insect toxin with no effect on mice. © 2017 The Protein Society.

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

PubMed

Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

2013-06-01

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

PubMed

Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

2014-02-18

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

PubMed Central

Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A.; Agu, Chukwuma A.; Wang, Xindan; Bernal, Juan A.; Sherratt, David J.; de la Cueva-Méndez, Guillermo

2014-01-01

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs. PMID:24449860

Deletion mutant comprising 198 residues of BoNT/A toxin receptor binding domain retained gt1b binding property but failed to induce protective antibody response in a mouse model.

PubMed

Singh, Manglesh Kumar; Gupta, Garima; Boopathi, Mannan; Gupta, Pallavi; Chauhan, Vinita; Tomar, Arvind; Singh, Lokendra; Dhaked, Ram Kumar

2012-05-01

The most effective protection against toxin is inducing protective immune response through vaccination that will produce neutralizing antibodies. Antibodies will bind to and clear toxin from the circulation before it can enter nerve cells and block neurotransmission and can also be used for development of detection system. In the present study we constructed a deletion mutant of the binding domain (1098-1296) to produce smallest toxin fragment as vaccine candidate against BoNT/A. The BoNT/A-HCC protein was highly expressed in Escherichia coli SG13009 and found to form inclusion bodies. The purified inclusion bodies were solubilized in 6 M guanidine-HCl containing 10 mM β-mercaptoethanol and 20 mM imidazole and the rBoNT/A-HCC was purified and refolded in a single step on Ni2+ affinity column. The purified protein was ∼98 % pure as assessed by SDS-polyacrylamide gel with the yield of 8 mg/L and showed binding to polysialoganglioside (GT1b). The rBoNT/A-HCC at dose of 40 μg/mouse generated high IgG antibody titre with predominance of IgG1 subtype, but failed to protect animals against BoNT/A challenge. Antibody titre in serum was determined by enzyme linked immunosorbent assay and specific binding to rBoNT/A-HCC was demonstrated by surface plasmon resonance (SPR), with a dissociation constant of 0.8 pM.

Isolation and characterization of α-elapitoxin-Bf1b, a postsynaptic neurotoxin from Malaysian Bungarus fasciatus venom.

PubMed

Rusmili, Muhamad Rusdi Ahmad; Tee, Ting Yee; Mustafa, Mohd Rais; Othman, Iekhsan; Hodgson, Wayne C

2014-03-15

Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom. Copyright © 2014 Elsevier Inc. All rights reserved.

Safety and immunogenicity of ricin vaccine, RVEc™, in a Phase 1 clinical trial.

PubMed

Pittman, Phillip R; Reisler, Ronald B; Lindsey, Changhong Y; Güereña, Fernando; Rivard, Robert; Clizbe, Denise P; Chambers, Matthew; Norris, Sarah; Smith, Leonard A

2015-12-16

Ricin is a potent toxin and potential bioterrorism weapon for which no specific licensed countermeasures are available. We report the safety and immunogenicity of the ricin vaccine RVEc™ in a Phase 1 (N=30) multiple-dose, open-label, non-placebo-controlled, dose-escalating (20, 50, and 100μg), single-center study. Each subject in the 20- and 50-μg dose groups (n=10 for each group) received three injections at 4-week intervals and was observed carefully for untoward effects of the vaccine; blood was drawn at predetermined intervals after each dose for up to 1 year. RVEc™ was safe and well tolerated at the 20- and 50-μg doses. The most common adverse events were pain at the injection site and headache. Of the 10 subjects who received a single 100-μg dose, two developed elevated creatine phosphokinase levels, which resolved without sequelae. No additional doses were administered to subjects in the 100-μg group. Immunogenicity of the vaccine was evaluated by measuring antibody response using the well standardized enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). Of the subjects in the 20- and 50-μg dose groups, 100% achieved ELISA anti-ricin IgG titers of 1:500 to 1:121,500 and 50% produced neutralizing anti-ricin antibodies measurable by TNA. Four subjects in the 50-μg group received a single booster dose of RVEc™ 20-21 months after the initial dose. The single booster was safe and well tolerated, resulting in no serious adverse events, and significantly enhanced immunogenicity of the vaccine in human subjects. Each booster recipient developed a robust anamnestic response with ELISA anti-ricin IgG titers of 1:13,500 to 1:121,500 and neutralizing antibody titers of 1:400 to 1:3200. Future studies will attempt to optimize dose, scheduling, and route of administration. This study is registered at clinicaltrials.gov (NCT01317667 and NCT01846104). Published by Elsevier Ltd.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

PubMed

Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

2018-06-01

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum , the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile . All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N -terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Electrophysiological response of chicken's jejunal epithelium to increasing levels of T-2 toxin.

PubMed

Yunus, Agha Waqar; Kröger, Susan; Tichy, Alexander; Zentek, Jürgen; Böhm, Josef

2013-02-01

The present investigations were conducted to test the effects of T-2 toxin on electrophysiological variables of jejunal epithelium of chicken. Jejunal segments of broilers were monitored in Ussing chambers in the presence of T-2 toxin at the levels of 0 (negative control), 0 (methanol/vehicle control), 0.1, 1, 5, and 10 μg/ml of buffer. T-2 toxin did not affect basal values of short circuit current (I(sc)), transmural potential difference, or tissue conductivity in the jejunal epithelium. T-2 toxin also did not statistically affect glucose-induced electrophysiological variables during the first 3 min of glucose induction. Compared to the vehicle control, the ouabain-sensitive I(sc) was negatively affected (P = 0.008) only under 5 μg of T-2 toxin/ml. Increasing levels of T-2 toxin negatively affected the ouabain-sensitive I(sc) in a cubic (P = 0.007) fashion. These data indicate that acute exposure to moderate levels of T-2 toxin may progressively impair the cation gradient across the jejunal epithelium.

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum.

PubMed

Vázquez-Iglesias, Lorena; Estefanell-Ucha, Borja; Barcia-Castro, Leticia; Páez de la Cadena, María; Álvarez-Chaver, Paula; Ayude-Vázquez, Daniel; Rodríguez-Berrocal, Francisco Javier

2017-01-01

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

PubMed Central

Estefanell-Ucha, Borja; Barcia-Castro, Leticia; Páez de la Cadena, María; Álvarez-Chaver, Paula; Ayude-Vázquez, Daniel; Rodríguez-Berrocal, Francisco Javier

2017-01-01

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot. PMID:28652930

OnabotulinumtoxinA therapy for compensatory hyperhidrosis.

PubMed

Adefusika, Jessica A; Brewer, Jerry D

2013-09-01

Compensatory hyperhidrosis due to the treatment for focal primary hyperhidrosis can be devastating and life-altering. The purpose of this paper is to discuss use of the iodine starch test and dilute botulinum toxin to decrease compensatory hyperhidrosis over large surface areas. A large area of the trunk affected by compensatory hyperhidrosis was treated with dilute botulinum toxin therapy using a starch iodine test for localization. The patient had exceptional results with his compensatory truncal sweating returning to normal and remaining stable for multiple months. Treating compensatory hyperhidrosis of large surface areas with dilute botulinum toxin therapy is effective and safe. © 2013 Wiley Periodicals, Inc.

Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

PubMed

Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

2015-12-23

Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

Vaccination with plasmid DNA encoding a mutant toxic shock syndrome toxin-1 ameliorates toxin-induced lethal shock in mice.

PubMed

Feng, Mao-Hui; Cui, Jing-Chun; Nakane, Akio; Hu, Dong-Liang

2013-09-01

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

Comparing ImmunoCard with two EIA assays for Clostridium difficile toxins.

PubMed

Chan, Edward L; Seales, Diane; Drum, Hong

2009-01-01

To compare three Clostridium difficile EIA kits for the detection of C. difficile toxins from clinical specimens. A total of 287 fresh and stored stool specimens were tested using all three assays. Stools with discrepant results were sent to a reference laboratory for tissue cytotoxin assay. Trinity Medical Center, a community hospital with network hospitals. Patients with diarrhea submitted stools for detection of C. difficile toxins. Of the 287 stool specimens, 116 were positive and 171 negative for C. difficile toxins. The sensitivity, specificity, and positive and negative predictive values of Meridian EIA assay were 99.1, 97.7, 96.6, and 99.4%; ImmunoCard were 100, 98.2, 97.5, and 100%; BioStar OIA assay were 94, 98.8, 98.2, and 96% respectively. ImmunoCardprovides the best sensitivity (100%) for C. difficile toxins A and B detection. The BioStar OIA rapid test missed seven positive stool specimens possibly due to failure to detect toxin B. ImmunoCard has slightly higher predictive values, shorter turnaround time and greater convenience compared to the Meridian EIA Assay. ImmunoCard may be cost effective not only in smaller laboratories, but also in high volume laboratories, when used on a STAT basis or single request.

Is there a relationship between the presence of the binary toxin genes in Clostridium difficile strains and the severity of C. difficile infection (CDI)?

PubMed

Berry, C E; Davies, K A; Owens, D W; Wilcox, M H

2017-12-01

Some strains of Clostridium difficile produce a binary toxin, in addition to the main C. difficile virulence factors (toxins A and B). There have been conflicting reports regarding the role of binary toxin and its relationship to the severity of C. difficile infection (CDI). Samples, isolates and clinical data were collected as part of a prospective multicentre diagnostic study. Clostridium difficile isolates (n = 1259) were tested by polymerase chain reaction (PCR) assay to detect binary toxin genes cdtA and cdtB. The PCR binary toxin gene results were compared with clinical severity and outcome data, including 30-day all-cause mortality. The 1259 isolates corresponded to 1083 different patients (October 2010 to September 2011). The prevalence of binary toxin positive strains was significantly higher in faecal samples with detectable toxin A/B than in those without toxin but that were positive by cytotoxigenic culture (26.3% vs. 10.3%, p < 0.001). The presence of binary toxin correlated moderately with markers of CDI severity (white cell count, serum albumin concentration and serum creatinine concentration). However, the risk ratio for all-cause mortality was 1.68 for binary toxin positive patients and patients were significantly less likely to survive if they had CDI caused by a binary toxin gene positive strain, even after adjusting for age (p < 0.001). The presence of binary toxin genes does not predict the clinical severity of CDI, but it is significantly associated with the risk of all-cause mortality.

Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

PubMed Central

Lee, Ki-Young; Lee, Bong-Jin

2016-01-01

Bacterial toxin–antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein–protein interactions. Accumulating knowledge about the structure–function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems. PMID:27782085

Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk.

PubMed

Wein, Lawrence M; Liu, Yifan

2005-07-12

We developed a mathematical model of a cows-to-consumers supply chain associated with a single milk-processing facility that is the victim of a deliberate release of botulinum toxin. Because centralized storage and processing lead to substantial dilution of the toxin, a minimum amount of toxin is required for the release to do damage. Irreducible uncertainties regarding the dose-response curve prevent us from quantifying the minimum effective release. However, if terrorists can obtain enough toxin, and this may well be possible, then rapid distribution and consumption result in several hundred thousand poisoned individuals if detection from early symptomatics is not timely. Timely and specific in-process testing has the potential to eliminate the threat of this scenario at a cost of <1 cent per gallon and should be pursued aggressively. Investigation of improving the toxin inactivation rate of heat pasteurization without sacrificing taste or nutrition is warranted.

Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuroki, T.; Chida, K.; Munakata, K.

1986-05-29

Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetra-decanoylphorbol-13-acetate. It also induced adenylate cyclase and through weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 ..mu..g 7,12-dimethyl-benz(a)anthracene, but no tumors developed. In the two-stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 ..mu..g) was appliedmore » twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas.« less

Effect of anthrax immune globulin on response to BioThrax (anthrax vaccine adsorbed) in New Zealand white rabbits.

PubMed

Malkevich, Nina V; Basu, Subhendu; Rudge, Thomas L; Clement, Kristin H; Chakrabarti, Ajoy C; Aimes, Ronald T; Nabors, Gary S; Skiadopoulos, Mario H; Ionin, Boris

2013-11-01

Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.

Addressing harmful algal blooms (HABs) impacts with ferrate(VI): Simultaneous removal of algal cells and toxins for drinking water treatment.

PubMed

Deng, Yang; Wu, Meiyin; Zhang, Huiqin; Zheng, Lei; Acosta, Yaritza; Hsu, Tsung-Ta D

2017-11-01

Although ferrate(VI) has long been recognized as a multi-purpose treatment agent, previous investigations regarding ferrate(VI) for addressing harmful algal blooms (HABs) impacts in drinking water treatment only focused on a single HAB pollutant (e.g. algal cells or algal toxins). Moreover, the performance of ferrate(VI)-driven coagulation was poorly investigated in comparison with ferrate(VI) oxidation, though it has been widely acknowledged as a major ferrate(VI) treatment mechanism. We herein reported ferrate(VI) as an emerging agent for simultaneous and effective removal of algal cells and toxins in a simulated HAB-impacted water. Ferrate(VI)-driven oxidation enabled algal cell inactivation and toxin decomposition. Subsequently, Fe(III) from ferrate(VI) reduction initiated an in-situ coagulation for cell aggregation. Cell viability (initial 4.26 × 10 4 cells/mL at pH 5.5 and 5.16 × 10 4 cells/mL at pH 7.5) decreased to 0.0% at ≥ 7 mg/L Fe(VI) at pH 5.5 and 7.5, respectively. Cell density and turbidity were dramatically decreased at pH 5.5 once ferrate(VI) doses were beyond their respective threshold levels, which are defined as minimum effective iron doses (MEIDs). However, the particulate removal at pH 7.5 was poor, likely because the coagulation was principally driven by charge neutralization and a higher pH could not sufficiently lower the particle surface charge. Meanwhile, algal toxins (i.e., microcystins) of 3.98 μg/L could be substantially decomposed at either pH. And the greater degradation achieved at pH 5.5 was due to the higher reactivity of ferrate(VI) at the lower pH. This study represents the first step toward the ferrate(VI) application as a promising approach for addressing multiple HABs impacts for water treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs

PubMed Central

Matoba, Nobuyuki; Magérus, Aude; Geyer, Brian C.; Zhang, Yunfang; Muralidharan, Mrinalini; Alfsen, Annette; Arntzen, Charles J.; Bomsel, Morgane; Mor, Tsafrir S.

2004-01-01

A vaccine that would engage the mucosal immune system against a broad range of HIV-1 subtypes and prevent epithelial transmission is highly desirable. Here we report fusing the mucosal targeting B subunit of cholera toxin to the conserved galactosylceramide-binding domain (including the ELDKWA-neutralizing epitope) of the HIV-1 gp41 envelope protein, which mediates the transcytosis of HIV-1 across the mucosal epithelia. Chimeric protein expressed in bacteria or plants assembled into oligomers that were capable of binding galactosyl-ceramide and GM1 gangliosides. Mucosal (intranasal) administration in mice of the purified chimeric protein followed by an i.p. boost resulted in transcytosis-neutralizing serum IgG and mucosal IgA responses and induced immunological memory. Plant production of mucosally targeted immunogens could be particularly useful for immunization programs in developing countries, where desirable product traits include low cost of manufacture, heat stability, and needle-free delivery. PMID:15347807

Effect of botulinum-A toxin to cremaster muscle: an experimental study.

PubMed

Cakmak, Murat; Cağlayan, Fatma; Erdemoğlu, Ali Kemal; Ulusoy, Sevgi

2003-10-01

A controversy exists on the definition, etiology and treatment of the retractile testes. In the present experimental study, we aimed to show the effect of botulinum-A toxin (Botox) on cremasteric muscle of a rat, and whether it may be an alternative to surgical treatment of retractile testis. Ten Wistar rats were used in the study. By stimulating cremasteric reflex, five compound muscle action potentials (CMAP) of the right and left cremasteric muscles of each rat were recorded using surface electrodes. Intramuscular injection of botulinum-A toxin was done to the right side. Saline was injected to the left cremasteric muscles, and the left side also served as control. CMAP of the cremasteric muscles were recorded 45 days after the injection. Statistical analysis was done using Wilcoxon Signed rank test. Mean CMAP of the right side was 3.25+/-1.39 microV before the injection and 0.44+/-0.25 microV after botulinum-A toxin injection. The difference was statistically significant (p<0.05). Mean CMAP on the left side was 3.48+/-0.32 microV and 3.14+/-1.12 microV at baseline and the end of the study, respectively. The difference was not statistically significant (p>0.05). The botulinum-A toxin paralyzes the cremasteric muscles of the rats. As cremasteric hypertonicity is accepted as one of the reasons for retractile testes, botulinum-A toxin injection to cremasteric muscles may be helpful in diagnosis and may be an alternative to surgical treatment of this pathology in repeated dosages. Long-term evaluation of this paralysis is necessary.

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

PubMed Central

Dubois, Thomas; Dancer-Thibonnier, Marie; Monot, Marc; Hamiot, Audrey; Bouillaut, Laurent; Soutourina, Olga; Martin-Verstraete, Isabelle

2016-01-01

The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. PMID:27297391

Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

PubMed Central

Salha, Danielle; Szeto, Jason; Myers, Lisa; Claus, Carol; Sheung, Anthony; Tang, Mei; Ljutic, Belma; Hanwell, David; Ogilvie, Karen; Ming, Marin; Messham, Benjamin; van den Dobbelsteen, Germie; Hopfer, Robert; Ochs, Martina M.

2012-01-01

Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia. PMID:22473606

Neutralizing antibodies elicited by a novel detoxified pneumolysin derivative, PlyD1, provide protection against both pneumococcal infection and lung injury.

PubMed

Salha, Danielle; Szeto, Jason; Myers, Lisa; Claus, Carol; Sheung, Anthony; Tang, Mei; Ljutic, Belma; Hanwell, David; Ogilvie, Karen; Ming, Marin; Messham, Benjamin; van den Dobbelsteen, Germie; Hopfer, Robert; Ochs, Martina M; Gallichan, Scott

2012-06-01

Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

PubMed Central

Gagnaire, Julie; Dauwalder, Olivier; Boisset, Sandrine; Khau, David; Freydière, Anne-Marie; Ader, Florence; Bes, Michèle; Lina, Gerard; Tristan, Anne; Reverdy, Marie-Elisabeth; Marchand, Adrienne; Geissmann, Thomas; Benito, Yvonne; Durand, Géraldine; Charrier, Jean-Philippe; Etienne, Jerome; Welker, Martin; Van Belkum, Alex; Vandenesch, François

2012-01-01

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01–10.24; p = 0.023; CI 95%: 1.20–12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies. PMID:22792394

Prevalence and Characterization of a Binary Toxin (Actin-Specific ADP-Ribosyltransferase) from Clostridium difficile

PubMed Central

Gonçalves, Carina; Decré, Dominique; Barbut, Frédéric; Burghoffer, Béatrice; Petit, Jean-Claude

2004-01-01

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated. PMID:15131151

Potential shortfall of pyramided transgenic cotton for insect resistance management

PubMed Central

Brévault, Thierry; Heuberger, Shannon; Zhang, Min; Ellers-Kirk, Christa; Ni, Xinzhi; Masson, Luke; Li, Xianchiun; Tabashnik, Bruce E.; Carrière, Yves

2013-01-01

To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the “pyramid” strategy uses plants that produce two or more toxins that kill the same pest. In the United States, this strategy has been adopted widely, with two-toxin Bt cotton replacing one-toxin Bt cotton. Although two-toxin plants are likely to be more durable than one-toxin plants, the extent of this advantage depends on several conditions. One key assumption favoring success of two-toxin plants is that they kill insects selected for resistance to one toxin, which is called “redundant killing.” Here we tested this assumption for a major pest, Helicoverpa zea, on transgenic cotton producing Bt toxins Cry1Ac and Cry2Ab. Selection with Cry1Ac increased survival on two-toxin cotton, which contradicts the assumption. The concentration of Cry1Ac and Cry2Ab declined during the growing season, which would tend to exacerbate this problem. Furthermore, analysis of results from 21 selection experiments with eight species of lepidopteran pests indicates that some cross-resistance typically occurs between Cry1A and Cry2A toxins. Incorporation of empirical data into simulation models shows that the observed deviations from ideal conditions could greatly reduce the benefits of the pyramid strategy for pests like H. zea, which have inherently low susceptibility to Bt toxins and have been exposed extensively to one of the toxins in the pyramid before two-toxin plants are adopted. For such pests, the pyramid strategy could be improved by incorporating empirical data on deviations from ideal assumptions about redundant killing and cross-resistance. PMID:23530245

Determination of the median toxic dose of type C botulism in lactating dairy cows

USGS Publications Warehouse

Moeller, R.B.; Puschner, B.; Walker, R.L.; Rocke, Tonie E.; Galey, F.D.; Cullor, J.S.; Ardans, A.A.

2003-01-01

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Degradation of pesticides with RSDL® (reactive skin decontamination lotion kit) lotion: LC-MS investigation.

PubMed

Fentabil, Messele; Gebremedhin, Mulu; Purdon, J Garfield; Cochrane, Laura; Goldman, Virginia Streusand

2018-09-01

This study examined the degradation of organophosphate (OP) and carbamate pesticides using RSDL ® (Reactive Skin Decontamination Lotion Kit) lotion. Degradation occurs from a nucleophilic substitution (SN) reaction between an ingredient in the RSDL lotion, potassium 2,3-butanedione monoximate (KBDO), with susceptible sites in the pesticides. Evaluation at several molar ratios of KBDO:test articles using liquid chromatography-mass spectrometry (LC-MS) techniques was performed. The OP test articles, parathion, paraoxon, parathion-methyl, paraoxon-methyl and chlorpyrifos were effectively degraded at molar ratios of four and above in less than 6min contact time. Malathion and malaoxon were similarly converted to inactive by-products at molar ratios as low as two in less than 4min. A minimum molar ratio of nine was found to be effective against the carbamate pesticide carbofuran. In the case of aldicarb, complete destruction was achieved at a molar ratio of fifteen and a reaction time of one hour. It is important to note that these studies are based on a direct liquid phase RSDL lotion reaction with the toxic chemicals without the added physical removal decontamination efficacy component provided by the sponge component of the RSDL kit. The RSDL kit is intended to be used to remove or neutralize chemical warfare agents (CWA) and T-2 toxin from the skin. In actual use, the majority of the CWA decontamination occurs through the combined action of the sponge in both removing the chemical from the skin, and in rapidly mixing the chemicals at a high molar ratio of KBDO:CWA within the pores of the sponge to enhance rapid neutralization of the chemical. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of Performance and Potential Clinical Impact of ProSpecT Shiga Toxin Escherichia coli Microplate Assay for Detection of Shiga Toxin-Producing E. coli in Stool Samples

PubMed Central

Gavin, Patrick J.; Peterson, Lance R.; Pasquariello, Anna C.; Blackburn, Joanna; Hamming, Mark G.; Kuo, Kuo J.; Thomson, Richard B.

2004-01-01

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing. PMID:15071021

Interaction of Type IV Toxin/Antitoxin Systems in Cryptic Prophages of Escherichia coli K-12.

PubMed

Wen, Zhongling; Wang, Pengxia; Sun, Chenglong; Guo, Yunxue; Wang, Xiaoxue

2017-03-01

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of "lemon-shaped" cells. Prolonged overproduction of YkfI led to the formation of "gourd-shaped" cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli , but ectopic expression in Pseudomonas and Shewanella did not cause the formation of "lemon-shaped" cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli .

Interaction of Type IV Toxin/Antitoxin Systems in Cryptic Prophages of Escherichia coli K-12

PubMed Central

Wen, Zhongling; Wang, Pengxia; Sun, Chenglong; Guo, Yunxue; Wang, Xiaoxue

2017-01-01

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of “lemon-shaped” cells. Prolonged overproduction of YkfI led to the formation of “gourd-shaped” cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli, but ectopic expression in Pseudomonas and Shewanella did not cause the formation of “lemon-shaped” cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli. PMID:28257056

Clinical and biochemical significance of toxin production by Aeromonas hydrophila.

PubMed Central

Kindschuh, M; Pickering, L K; Cleary, T G; Ruiz-Palacios, G

1987-01-01

Production of cytotoxin and enterotoxin by Aeromonas strains obtained from stools of 50 children in Mexico and Texas and from blood of 9 children with sepsis was determined. Results were correlated with clinical features of infected children as well as with biochemical traits of Aeromonas strains. Cytotoxin was produced by 40 of 42 Aeromonas strains (95%) isolated from stools of children with diarrhea, by all 8 isolates from stools of well children, and by all 9 isolates from children with sepsis. There was no difference in the quantities (amount of cytotoxin per milligram of protein required to kill 50% of the cells) of cytotoxin produced and in clinical manifestations among the groups. None of the isolates produced a toxin that could be neutralized by antiserum raised against Shiga toxin produced by Shigella dysenteriae 1 60R. Heat-labile-like enterotoxin (LT) was produced by 26 of 42 stool isolates (62%), while only 1 of the 42 isolates (2%) produced enterotoxinlike activity in suckling mice; 65% of the cytotoxin-producing strains also produced an LT-like material. All strains from blood produced LT-like material, and 2 of 6 (33%) produced activity in suckling mice. All strains produced hemolysin; 37 of 57 (65%) were Voges-Proskauer positive; 27 of 57 (47%) were lysine decarboxylase positive by API 20E strips, none were positive for lysine decarboxylose production by lysin-iron agar slants at 24 h, but 17 of 54 (31%) were positive at 48 h. There was no correlation between biochemical reactions and enterotoxin or cytotoxin production. There appears to be no correlation between toxin production by Aeromonas spp. and gastroenteritis. PMID:3584426

Immuno-therapy of Acute Radiation Syndromes : Extracorporeal Immuno-Lympho-Plasmo-Sorption.

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Slava

Methods Results Summary and conclusions Introduction: Existing Medical Management of the Acute Radiation Syndromes (ARS) does not include methods of specific immunotherapy and active detoxication. Though the Acute Radiation Syndromes were defined as an acute toxic poisonous with development of pathological processes: Systemic Inflammatory Response Syndrome (SIRS), Toxic Multiple Organ Injury (TMOI), Toxic Multiple Organ Dysfunction Syndrome(TMODS), Toxic Multiple Organ Failure (TMOF). Radiation Toxins of SRD Group play an important role as the trigger mechanisms in development of the ARS clinical symptoms. Methods: Immuno-Lympho-Plasmo-Sorption is a type of Immuno-therapy which includes prin-ciples of immunochromato-graphy, plasmopheresis, and hemodialysis. Specific Antiradiation Antitoxic Antibodies are the active pharmacological agents of immunotherapy . Antiradia-tion Antitoxic Antibodies bind selectively to Radiation Neurotoxins, Cytotoxins, Hematotox-ins and neutralize their toxic activity. We have developed the highly sensitive method and system for extracorporeal-immune-lypmh-plasmo-sorption with antigen-specific IgG which is clinically important for treatment of the toxic and immunologic phases of the ARS. The method of extracorporeal-immune-lypmh-plasmo-sorption includes Antiradiation Antitoxic Antibodies (AAA) immobilized on microporous polymeric membranes with a pore size that is capable to provide diffusion of blood-lymph plasma. Plasma of blood or lymph of irradiated mammals contains Radiation Toxins (RT) that have toxic and antigenic properties. Radiation Toxins are Antigen-specific to Antitoxic blocking antibodies (Immunoglobulin G). Plasma diffuses through membranes with immobilized AAA and AA-antibodies bind to the polysaccharide chain of tox-ins molecules and complexes of AAA-RT that are captured on membrane surfaces. RT were removed from plasma. Re-transfusion of plasma of blood and lymph had been provided. We show a statistical significant reduction in postradiation lethality.

Isolation and characterization of a presynaptic neurotoxin, P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom.

PubMed

Rusmili, Muhamad Rusdi Ahmad; Yee, Tee Ting; Mustafa, Mohd Rais; Hodgson, Wayne C; Othman, Iekhsan

2014-10-01

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, β-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of β-bungarotoxin in this species was due to divergence during evolution prior to evolution of β-bungarotoxin. In this study, we have isolated a β-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A2 subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique β-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of sodium channel activators by a rapid fluorimetric microplate assay.

PubMed

Louzao, M C; Vieytes, M R; Yasumoto, T; Botana, L M

2004-04-01

Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, time-consuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i). the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potential-dependent, and (ii). dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.

Toxins vapC and pasB from prokaryotic TA modules remain active in mammalian cancer cells.

PubMed

Wieteska, Łukasz; Skulimowski, Aleksander; Cybula, Magdalena; Szemraj, Janusz

2014-09-30

Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

PubMed

Bever, Candace S; Barnych, Bogdan; Hnasko, Robert; Cheng, Luisa W; Stanker, Larry H

2018-06-28

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides . The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides .

A relationship between avian carcasses and living invertebrates in the epizootiology of avian botulism

USGS Publications Warehouse

Duncan, Ruth M.; Jensen, Wayne I.

1976-01-01

A survey of the sources of Clostridium botulinum type C toxin possibly utilized as food by aquatic birds in an epizootic area of avian botulism in northern Utah showed that living aquatic and terrestrial invertebrates normally found in close association with dead, decomposing birds commonly carried the toxin. Of 461 samples associated with 21 species of avian carcasses, 198 were toxin-positive. Invertebrate species not normally scavengers of vertebrate tissues were less commonly and less highly toxic, particularly when captured 30 cm or more from a carcass; six of 237 samples of such aquatic invertebrates contained low-level toxin. Of the species tested, blow fly larvae (Calliphoridae) were the most consistently and highly toxic, although others, particularly adult and larval stages of several species of beetles (Coleoptera), contained toxin at levels probably significant in the epizootiology of the disease. An estimated 0.05 to 0.25 g of the most toxic fly larvae or 15 g of the most toxic beetles tested carried a mediam lethal dose for an adult mallard duck. Examination of stomach contents of aquatic birds dead of botulism showed that some had consumed invertebrates.

Comparison of Diagnostic Algorithms for Detecting Toxigenic Clostridium difficile in Routine Practice at a Tertiary Referral Hospital in Korea.

PubMed

Moon, Hee-Won; Kim, Hyeong Nyeon; Hur, Mina; Shim, Hee Sook; Kim, Heejung; Yun, Yeo-Min

2016-01-01

Since every single test has some limitations for detecting toxigenic Clostridium difficile, multistep algorithms are recommended. This study aimed to compare the current, representative diagnostic algorithms for detecting toxigenic C. difficile, using VIDAS C. difficile toxin A&B (toxin ELFA), VIDAS C. difficile GDH (GDH ELFA, bioMérieux, Marcy-l'Etoile, France), and Xpert C. difficile (Cepheid, Sunnyvale, California, USA). In 271 consecutive stool samples, toxigenic culture, toxin ELFA, GDH ELFA, and Xpert C. difficile were performed. We simulated two algorithms: screening by GDH ELFA and confirmation by Xpert C. difficile (GDH + Xpert) and combined algorithm of GDH ELFA, toxin ELFA, and Xpert C. difficile (GDH + Toxin + Xpert). The performance of each assay and algorithm was assessed. The agreement of Xpert C. difficile and two algorithms (GDH + Xpert and GDH+ Toxin + Xpert) with toxigenic culture were strong (Kappa, 0.848, 0.857, and 0.868, respectively). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of algorithms (GDH + Xpert and GDH + Toxin + Xpert) were 96.7%, 95.8%, 85.0%, 98.1%, and 94.5%, 95.8%, 82.3%, 98.5%, respectively. There were no significant differences between Xpert C. difficile and two algorithms in sensitivity, specificity, PPV and NPV. The performances of both algorithms for detecting toxigenic C. difficile were comparable to that of Xpert C. difficile. Either algorithm would be useful in clinical laboratories and can be optimized in the diagnostic workflow of C. difficile depending on costs, test volume, and clinical needs.

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

PubMed Central

Tabashnik, Bruce E.; Fabrick, Jeffrey A.; Unnithan, Gopalan C.; Yelich, Alex J.; Masson, Luke; Zhang, Jie; Bravo, Alejandra; Soberón, Mario

2013-01-01

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. PMID:24244692

HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea.

PubMed

Jørgensen, Mikkel G; Pandey, Deo P; Jaskolska, Milena; Gerdes, Kenn

2009-02-01

Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

Evaluation of different immuno-magnetic beads and selective plating media for the confirmation of Shiga toxin-producing Escherichia coli detected by molecular methods in beef trim enrichment broths

USDA-ARS?s Scientific Manuscript database

Introduction: Shiga toxin-producing E. coli of serogroups O26, O45, O103, O111, O121 and O145 are a testing concern for the beef industry and regulators. Numerous tests are available that attempt to predict the presence of these pathogens. However, potential positive results require culture confir...

Toxin content and cytotoxicity of algal dietary supplements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heussner, A.H.; Mazija, L.; Fastner, J.

Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earliermore » market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC–MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤ 1 μg MC-LR equivalents g{sup −1} dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable. -- Highlights: ► Marketed algae dietary supplements were analyzed for toxins. ► Methods: Phosphatase inhibition assay (PPIA), Adda-ELISA, LC-MS/MS. ► Aph. flos-aquae products all tested positive for microcystins. ► Products tested negative for nodularins, saxitoxins, anatoxin-a, cylindrospermopsin. ► Extracts from all products were cytotoxic.« less

A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

PubMed

Smith, Andrew B; López-Villarejo, Juan; Diago-Navarro, Elizabeth; Mitchenall, Lesley A; Barendregt, Arjan; Heck, Albert J; Lemonnier, Marc; Maxwell, Anthony; Díaz-Orejas, Ramón

2012-01-01

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

Method Development for Comprehensive Extraction and Analysis of Marine Toxins: Liquid-Liquid Extraction and Tandem Liquid Chromatography Separations Coupled to Electrospray Tandem Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wunschel, David S.; Valenzuela, Blandina R.; Kaiser, Brooke L. Deatherage

A variety of toxins are produced by marine and freshwater microorganisms that present a threat to human health. These toxins have diverse chemical properties and specifically, a range of hydrophobicity. Methods for extraction and identification of these toxins are often geared toward specific classes of toxin depending on the sample type. There is a need for a general method of toxin extraction and identification for screening samples where the likely toxin content is not known a priori. Here, we have applied a general method for metabolite extraction to toxin containing samples. This method was coupled with a simple dual liquidmore » chromatography approach for separating a broad range of toxins. This liquid chromatography approach was coupled to triple quadrupole and quadrupole time-of-flight MS/MS platforms. The method was testing on a fish matrix for recovery of palytoxin as well as marine corals for detection of natural mixtures of palytoxin analogues. The recovery of palytoxin was found to produce a linear response (R 2 of 0.95) when spiked into the fish matrix with a limit of quantitation of 2.5 ng/μL and recovery efficiency of 73% +/- 9%. The screening of corals revealed varying amount of palytoxin, and in one case, different palytoxin structural analogues. This demonstration illustrates the potential utility of this method for toxin extraction and detection.« less

Method Development for Comprehensive Extraction and Analysis of Marine Toxins: Liquid-Liquid Extraction and Tandem Liquid Chromatography Separations Coupled to Electrospray Tandem Mass Spectrometry

DOE PAGES

Wunschel, David S.; Valenzuela, Blandina R.; Kaiser, Brooke L. Deatherage; ...

2018-05-09

A variety of toxins are produced by marine and freshwater microorganisms that present a threat to human health. These toxins have diverse chemical properties and specifically, a range of hydrophobicity. Methods for extraction and identification of these toxins are often geared toward specific classes of toxin depending on the sample type. There is a need for a general method of toxin extraction and identification for screening samples where the likely toxin content is not known a priori. Here, we have applied a general method for metabolite extraction to toxin containing samples. This method was coupled with a simple dual liquidmore » chromatography approach for separating a broad range of toxins. This liquid chromatography approach was coupled to triple quadrupole and quadrupole time-of-flight MS/MS platforms. The method was testing on a fish matrix for recovery of palytoxin as well as marine corals for detection of natural mixtures of palytoxin analogues. The recovery of palytoxin was found to produce a linear response (R 2 of 0.95) when spiked into the fish matrix with a limit of quantitation of 2.5 ng/μL and recovery efficiency of 73% +/- 9%. The screening of corals revealed varying amount of palytoxin, and in one case, different palytoxin structural analogues. This demonstration illustrates the potential utility of this method for toxin extraction and detection.« less

A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions

PubMed Central

Mitchenall, Lesley A.; Barendregt, Arjan; Heck, Albert J.; Lemonnier, Marc; Maxwell, Anthony; Díaz-Orejas, Ramón

2012-01-01

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets. PMID:23029540

Recombinant vaccine displaying the loop-neutralizing determinant from protective antigen completely protects rabbits from experimental inhalation anthrax.

PubMed

Oscherwitz, Jon; Yu, Fen; Jacobs, Jana L; Cease, Kemp B

2013-03-01

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2β2-2β3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD(50)) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.

Sensitive assays enable detection of serum IgG antibodies against Clostridium difficile toxin A and toxin B in healthy subjects and patients with Clostridium difficile infection.

PubMed

Zhao, Xuemei; Bender, Florent; Shukla, Rajiv; Kang, John J; Caro-Aguilar, Ivette; Laterza, Omar F

2016-04-01

Pathogenic Clostridium difficile produces two proinflammatory exotoxins, toxin A and toxin B. Low level of serum antitoxin IgG antibodies is a risk factor for the development of primary and recurrent C. difficile infection (CDI). We developed and validated two sensitive, titer-based electrochemiluminescence assays for the detection of serum antibody levels against C. difficile toxins A and B. These assays demonstrated excellent precision. The sensitivity of the assays allowed the detection of antitoxin A and antitoxin B IgG antibodies in all tested serum samples during assay validation. The validated titer-based assays enable assessment of antitoxin A and antitoxin B IgG antibodies as potential biomarkers to identify patients with CDI at increased risk for CDI recurrence.

Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

PubMed

Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

2013-06-15

The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antimicrobial Nanoparticle for the Treatment of Bacterial Infection

NASA Astrophysics Data System (ADS)

Pornpattananangkul, Dissaya

Liposomes are spherical lipid vesicles with bilayered membrane structure, which have been recognized as one of the most widely used carriers for delivering a myriad of pharmaceuticals. Liposomes can carry both hydrophilic and hydrophobic agents with high efficiency and protect them from undesired effects of external conditions. However, the applications of liposomes are usually limited by their instability during storage. They are inclined to fuse with one another immediately after preparation, resulting in undesired mixing, increase in size, and payload loss. To overcome this limitation, this dissertation will focus on the technology to stabilize liposomes during storage and destabilize at specific conditions in order to allow controllable therapeutic release, as well as demonstrate their application to treat one of the bacterial infection diseases, acne vulgaris. The first area of this research is stimuli-responsive liposomes development, where the liposomes are stabilized by introducing gold nanoparticles to adsorb to their surface. As a result, the liposomes are prevented from fusing with one another and undesirable payload release during storage or physiological environments. Moreover, therapeutic is controllably released depending on environment conditions, such as acidic pH and bacterial virulence factor. In case of acid-responsive liposomes, the bound gold nanoparticles can effectively prevent liposomes from fusing with one another at neutral pH value, while at acidic environment (e.g. pH<5), the gold particle stabilizers will fall off from the liposomes, thereby reinstalling the fusion activity of liposomes. The fusion activity of the stabilized liposomes is found to be 25% at pH=7, in contrast to 80% at pH=4. Another stimulus that can activate drug release from liposomes is virulence factor released from bacteria themselves, such as bacterial toxin. When nanoparticle-stabilized liposomes encounter with bacteria that secrete toxin, the toxin will insert into the liposome membranes and form pores, through which the encapsulated therapeutic agents are released. The released drugs subsequently impose antimicrobial effects on the toxin-secreting bacteria. It was observed that in the presence of toxin-secreting bacteria, 100% of the encapsulated antibiotics were released from the gold nanoparticle-stabilized liposomes and bacterial growth was effectively inhibited by the released antibiotics in 24 h. The second area is to demonstrate an application of the invented technology to treat acne vulgaris by delivering therapeutics to the acne-causing bacteria, named Propionibacterium acnes (P.acnes). First, lauric acid (LA), an antimicrobial with strong activity against P. acnes, is encapsulated in liposomes (LipoLA), which is shown to effectively kill the bacteria by fusion with the bacterial membrane, resulting in a direct insertion of LA molecules to the membrane and destruction of its surface structure in vitro and in vivo. The system is then further improved by the acid-responsive technology based on the fact that the acne lesions on human skin are typically acidic. Demonstrated by fluorescent and antimicrobial experiments, the bound gold nanoparticles effectively prevent LipoLA from fusing with one another at neutral pH value. However, at acidic condition, the gold particles detatch from LipoLA surface, allowing the fusion with P.acnes membrane and lauric acid delivery, resulting in a complete killing effect. The stimuli-responsive liposomes presented here provide a new, safe, and effective approach to treat bacterial infections. They can be broadly applied to treat a variety of infections caused by bacteria that reside in acidic environment and secrete pore-forming toxins.

Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels

PubMed Central

Salari, Autoosa; Vega, Benjamin S.; Milescu, Lorin S.; Milescu, Mirela

2016-01-01

Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

PubMed

Meza-Lucas, Antonio; Pérez-Villagómez, María-Fernanda; Martínez-López, José-Patricio; García-Rodea, Ricardo; Martínez-Castelán, María-Guadalupe; Escobar-Gutiérrez, Alejandro; de-la-Rosa-Arana, Jorge-Luis; Villanueva-Zamudio, Altagracia

2016-09-01

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves.

PubMed

Valgaeren, Bonnie R; Pardon, Bart; Goossens, Evy; Verherstraeten, Stefanie; Roelandt, Sophie; Timbermont, Leen; Van Der Vekens, Nicky; Stuyvaert, Sabrina; Gille, Linde; Van Driessche, Laura; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip; Deprez, Piet

2015-07-10

Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin.

Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves

PubMed Central

Valgaeren, Bonnie R.; Pardon, Bart; Goossens, Evy; Verherstraeten, Stefanie; Roelandt, Sophie; Timbermont, Leen; Van Der Vekens, Nicky; Stuyvaert, Sabrina; Gille, Linde; Van Driessche, Laura; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip; Deprez, Piet

2015-01-01

Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin. PMID:26184311

Impact of toxigenic Clostridium difficile polymerase chain reaction testing on the clinical microbiology laboratory and inpatient epidemiology.

PubMed

Napierala, Maureen; Munson, Erik; Skonieczny, Patrice; Rodriguez, Sonia; Riederer, Nancy; Land, Gayle; Luzinski, Mary; Block, Denise; Hryciuk, Jeanne E

2013-08-01

Conversion from Clostridium difficile toxin A/B EIA to tcdB polymerase chain reaction for diagnosis of C. difficile infection (CDI) resulted in significant decreases in laboratory testing volume and largely unchanged C. difficile toxin detection rates. Decreases in healthcare-associated CDI rates (P ≤ 0.05) reflected a clinical practice benefit of this conversion. Copyright © 2013 Elsevier Inc. All rights reserved.

Glucosylation and other biotransformations of T-2 toxin by yeasts of the Trichomonascus clade

USDA-ARS?s Scientific Manuscript database

Twenty-five yeasts assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including three Trichomonascus species and 22 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert T-2 toxin, a Fusarium trichothecene mycotoxin, to less toxic product...

Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes, Micrurus altirostris and M. corallinus.

PubMed

Corrêa-Netto, Carlos; Junqueira-de-Azevedo, Inácio de L M; Silva, Débora A; Ho, Paulo L; Leitão-de-Araújo, Moema; Alves, Maria Lúcia M; Sanz, Libia; Foguel, Débora; Zingali, Russolina Benedeta; Calvete, Juan J

2011-08-24

The venom proteomes of Micrurus altirostris and M. corallinus were analyzed by combining snake venomics and venom gland transcriptomic surveys. In both coral snake species, 3FTx and PLA(2) were the most abundant and diversified toxin families. 33 different 3FTxs and 13 PLA(2) proteins, accounting respectively for 79.5% and 13.7% of the total proteins, were identified in the venom of M. altirostris. The venom of M. corallinus comprised 10 3FTx (81.7% of the venom proteome) and 4 (11.9%) PLA(2) molecules. Transcriptomic data provided the full-length amino acid sequences of 18 (M. altirostris) and 10 (M. corallinus) 3FTxs, and 3 (M. altirostris) and 1 (M. corallinus) novel PLA(2) sequences. In addition, venom from each species contained single members of minor toxin families: 3 common (PIII-SVMP, C-type lectin-like, L-amino acid oxidase) and 4 species-specific (CRISP, Kunitz-type inhibitor, lysosomal acid lipase in M. altirostris; serine proteinase in M. corallinus) toxin classes. The finding of a lipase (LIPA) in the venom proteome and in the venom gland transcriptome of M. altirostris supports the view of a recruitment event predating the divergence of Elapidae and Viperidae more than 60 Mya. The toxin profile of both M. altirostris and M. corallinus venoms points to 3FTxs and PLA(2) molecules as the major players of the envenoming process. In M. altirostris venom, all major, and most minor, 3FTxs display highest similarity to type I α-neurotoxins, suggesting that these postsynaptically acting toxins may play the predominant role in the neurotoxic effect leading to peripheral paralysis, respiratory arrest, and death. M. corallinus venom posesses both, type I α-neurotoxins and a high-abundance (26% of the venom proteome) protein of subfamily XIX of 3FTxs, exhibiting similarity to bucandin from Malayan krait, Bungarus candidus, venom, which enhances acetylcholine release presynaptically. This finding may explain the presynaptic neurotoxicity of M. corallinus venom and the lack of this effect in M. altirostris venom. The anti-Micrurus (corallinus and frontalis) antivenom produced by Instituto Butantan quantitatively immunodepleted the minor toxins from M. altirostris and M. corallinus venoms but showed impaired crossreactivity towards their major 3FTx and PLA(2) molecules. The structural diversity of 3FTxs among Micrurus sp. may underlay the impaired cross-immunoreactivity of the Butantan antivenom towards M. altirostris and M. corallinus toxins, hampering the possibility to raise an antivenom against a simple venom mixture exhibiting paraspecific neutralization of other Micrurus venoms. Copyright © 2011 Elsevier B.V. All rights reserved.

Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

PubMed

Bache, Christina; Spreitzer, Ingo; Becker, Bjoern; Loeschner, Bettina; Rosskopf, Ute; Hanschmann, Kay-Martin; Schwanig, Michael; Schneider, Christian K; Lieb, Bernhard; Montag, Thomas

2012-08-01

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.

Optimization of hydrophilic interaction liquid chromatography/mass spectrometry and development of solid-phase extraction for the determination of paralytic shellfish poisoning toxins.

PubMed

Turrell, Elizabeth; Stobo, Lesley; Lacaze, Jean-Pierre; Piletsky, Sergey; Piletska, Elena

2008-01-01

The combination of hydrophilic interaction liquid chromatography (HILIC) and liquid chromatography/mass spectrometry (LC/MS) for the determination of paralytic shellfish poisoning (PSP) toxins has been proposed for use in routine monitoring of shellfish. In this study, methods for the detection of multiple PSP toxins [saxitoxin (STX), neosaxitoxin (NEO), decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNEO), gonyautoxins 1-5 (GTX1, GTX2, GTX3, GTX4, GTX5), decarbamoyl gonyautoxins (dcGTX2 and dcGTX3), and the N-sulfocarbamoyl C toxins (C1 and C2)] were optimized using single (MS) and triple quadrupole (MS/MS) instruments. Chromatographic separation of the toxins was achieved by using a TSK-gel Amide-80 analytical column, although superior chromatography was observed through application of a ZIC-HILIC column. Preparative procedures used to clean up shellfish extracts and concentrate PSP toxins prior to analysis were investigated. The capacity of computationally designed polymeric (CDP) materials and HILIC solid-phase extraction (SPE) cartridges to retain highly polar PSP toxins was explored. Three CDP materials and 2 HILIC cartridges were assessed for the extraction of PSP toxins from aqueous solution. Screening of the CDPs showed that all tested polymers adsorbed PSP toxins. A variety of elution procedures were examined, with dilute 0.01% acetic acid providing optimum recovery from a CDP based on 2-(trifluoromethyl)acrylic acid as the monomer. ZIC-HILIC SPE cartridges were superior to the PolyLC equivalent, with recoveries ranging from 70 to 112% (ZIC-HILIC) and 0 to 90% (PolyLC) depending on the PSP toxin. It is proposed that optimized SPE and HILIC-MS methods can be applied for the quantitative determination of PSP toxins in shellfish.

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

PubMed

Rocha, Surza L G; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Angulo, Yamileth; Lomonte, Bruno; Valente, Richard H; Domont, Gilberto B; Perales, Jonas

2017-01-16

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA 2 , a few spots corresponding to acidic PLA 2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. Local tissue damage induced by myotoxic PLA 2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein. Copyright © 2016. Published by Elsevier B.V.

The epizootiology of type C botulism in fish-eating birds at Salton Sea, California

USGS Publications Warehouse

Nol, P.

2002-01-01

During 1996, type C avian botulism killed over 15,000 fish-eating birds at the Salton Sea in southern California. Amont those affected were nearly 10,000 western white pelicans (Pelecanus erythrorhynchos) and over 1,200 endangered California brown pelicans (Pelecanus occidentalis californicus). Since 1996, smaller epizootics have occurred every year. Type C botulism is not typically associated with fish-eating birds. In the case of the Salton Sea, Mozambique tilapia (Oreochromis mossambicus) are the suspected source of type C toxin, although the mechanism by which the fish acquire the toxin is still unknown. The goals of this study were to: 1) Determine presence/absence of active Clostridium botulinum type C and type C botulinum toxin in tilapia in the Salton Sea. 2) Use geospatial analyses to evaluate relationships between patterns of mortality in birds and fish and presence/absence of toxin and/or toxin-producing bacteria in sediments and fish. We investigated a method of detecting C. botulinum type C cells in the intestinal contents of Mozambique tilapia. This method involved extraction of predominantly cellular DNA and uses a polymerase chain reaction assay to detect presence of type C toxin gene. We collected sick, dead and healthy fish from various sites throughout the Sea during the summers of 1999 to 2001 in order to test them for the presence of active C. botulinum type C by PCR and for the presence of type C toxin by ELISA and mouse test. The results demonstrate that the tilapia population in the Salton Sea harbors C. botulinum type C cells within their gastrointestinal tract and the prevalence of this organism varies from year to year. The total number of fish with toxin-producing bacteria was significantly greater in 2000 than in 2001. No difference in the numbers of positives was detected between sick and dead fish compared to live fish, and there were no differences noted with regard to location of fish collection. The prevalence of active type C toxin in tilapia was low and the majority of samples positive for toxin by ELISA were negative on mouse test. Further investigation is currently underway. From 1999-2001, routine surveillance was conducted July through November to detect morbidity and mortality in pelicans. Location of retrieval and total number of birds collected were documented. In all three years, the majority of botulism-affected pelicans were retrieved from the vicinities of the river deltas although the Whitewater River had the fewest birds of the three. It has yet to be determined what roles the tilapia and pelican populations play in influencing these patterns.

Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

PubMed Central

Gharamah, Abdullah A; Moharram, Ahmed M; Ismail, Mady A; AL-Hussaini, Ashraf K

2014-01-01

Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2), sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin). Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss. PMID:24008795

Novel Covariance-Based Neutrality Test of Time-Series Data Reveals Asymmetries in Ecological and Economic Systems

PubMed Central

Burby, Joshua W.; Lacker, Daniel

2016-01-01

Systems as diverse as the interacting species in a community, alleles at a genetic locus, and companies in a market are characterized by competition (over resources, space, capital, etc) and adaptation. Neutral theory, built around the hypothesis that individual performance is independent of group membership, has found utility across the disciplines of ecology, population genetics, and economics, both because of the success of the neutral hypothesis in predicting system properties and because deviations from these predictions provide information about the underlying dynamics. However, most tests of neutrality are weak, based on static system properties such as species-abundance distributions or the number of singletons in a sample. Time-series data provide a window onto a system’s dynamics, and should furnish tests of the neutral hypothesis that are more powerful to detect deviations from neutrality and more informative about to the type of competitive asymmetry that drives the deviation. Here, we present a neutrality test for time-series data. We apply this test to several microbial time-series and financial time-series and find that most of these systems are not neutral. Our test isolates the covariance structure of neutral competition, thus facilitating further exploration of the nature of asymmetry in the covariance structure of competitive systems. Much like neutrality tests from population genetics that use relative abundance distributions have enabled researchers to scan entire genomes for genes under selection, we anticipate our time-series test will be useful for quick significance tests of neutrality across a range of ecological, economic, and sociological systems for which time-series data are available. Future work can use our test to categorize and compare the dynamic fingerprints of particular competitive asymmetries (frequency dependence, volatility smiles, etc) to improve forecasting and management of complex adaptive systems. PMID:27689714

Vaccination with nontoxic mutant toxic shock syndrome toxin 1 protects against Staphylococcus aureus infection.

PubMed

Hu, Dong-Liang; Omoe, Katsuhiko; Sasaki, Sanae; Sashinami, Hiroshi; Sakuraba, Hirotake; Yokomizo, Yuichi; Shinagawa, Kunihiro; Nakane, Akio

2003-09-01

To investigate whether vaccination with nontoxic mutant toxic shock syndrome toxin 1 (mTSST-1) can protect against Staphylococcus aureus infection, mice were vaccinated with mTSST-1 and challenged with viable S. aureus. Survival in the mTSST-1-vaccinated group was higher, and bacterial counts in organs were significantly lower than those of control mice. Passive transfer of mTSST-1-specific antibodies also provided protection against S. aureus-induced septic death. Interferon (IFN)-gamma production in the serum samples and spleens from vaccinated mice was significantly decreased compared with that in controls, whereas interleukin-10 titers were significantly higher in vaccinated mice. IFN-gamma and tumor necrosis factor-alpha production in vitro were significantly inhibited by serum samples from mTSST-1-immunized mice but not from control mice. These results suggest that vaccination with mTSST-1 devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibodies as well as by the down-regulation of IFN-gamma production.

Acid-triggered membrane insertion of Pseudomonas exotoxin A involves an original mechanism based on pH-regulated tryptophan exposure.

PubMed

Méré, Jocelyn; Morlon-Guyot, Juliette; Bonhoure, Anne; Chiche, Laurent; Beaumelle, Bruno

2005-06-03

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes

PubMed Central

Mazor, Ronit; Eberle, Jaime A.; Hu, Xiaobo; Vassall, Aaron N.; Onda, Masanori; Beers, Richard; Lee, Elizabeth C.; Kreitman, Robert J.; Lee, Byungkook; Baker, David; King, Chris; Hassan, Raffit; Benhar, Itai; Pastan, Ira

2014-01-01

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins. PMID:24799704

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

PubMed

Ivie, Susan E; McClain, Mark S

2012-09-25

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

PubMed Central

Ivie, Susan E.; McClain, Mark S.

2012-01-01

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

LC and LD50 values of Bacillus thuringiensis Serovar japonensis strain buibui toxin to Oriental beetle and northern masked chafer larvae (Coleoptera: Scarabaeidae).

PubMed

Mashtoly, Tamer A; El-Zemaity, Mohamed El-Said; Hussien, Mohamed I; Alm, Steven R

2009-10-01

Bacillus thuringiensis serovar japonensis strain Buibui has the potential to be an important control agent for pest scarabs. Bioassays were designed to test B. t. japonensis against two of the major turf and ornamental scarab pests infesting turfgrasses and ornamentals and to serve as a basis for further tests against other scarab pests. LC and LD50 values of B. t. serovarjaponensis strain Buibui toxin and spores were determined by four different bioassays for the oriental beetle, Anomala orientalis (Waterhouse), and northern masked chafer, Cyclocephala borealis Arrow. Oriental beetle larvae were bioassayed in autoclaved and nonautoclaved soil from where they were collected (Kingston, RI [native]), in nonautoclaved soil from where the northern masked chafer larvae were collected (Groton, CT [foreign]), and per os. Northern masked chafer larvae were bioassayed in autoclaved and nonautoclaved soil from where they were collected (Groton, CT [native]), in nonautoclaved soil from where the oriental beetle larvae were collected (Kingston, RI [foreign]) and per os. LC50 values of 3.93 microg toxin/g autoclaved native soil, 1.80 microg toxin/g nonautoclaved native soil, and 0.42 microg toxin/g nonautoclaved foreign soil and an LD50 value of 0.41 microg per os were determined at 14 d forA. orientalis. LC50 values of 588.28 microg toxin/g autoclaved native soil, 155.10 microg toxin/g nonautoclaved native soil, 265.32 microg toxin/g nonautoclaved foreign soil, and LD50 of 5.21 microg per os were determined at 14 d (soils) and 10 d (per os) for C. borealis. There were significant differences in LC50 values for oriental beetles in autoclaved, nonautoclaved native soil and nonautoclaved foreign soil. There were significant differences in LCo values for northern masked chafers in autoclaved and nonautoclaved native soil. B. t. japonensis can be applied now for control of oriental beetles at rates that are economically competitive with synthetic chemicals. If we can determine the component of nonautoclaved soil that enhances the activity of toxin, it may be possible to lower the rates of toxin needed for control to more economical levels for more difficult to control species such as the northern masked chafer.

Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation.

PubMed

Ayari-Riabi, Sana; Trimaille, Thomas; Mabrouk, Kamel; Bertin, Denis; Gigmes, Didier; Benlasfar, Zakaria; Zaghmi, Ahlem; Bouhaouala-Zahar, Balkiss; Elayeb, Mohamed

2016-04-04

Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (D,L-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50. Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575 μg/kg and 1300 μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875 μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258 μg/mL and 186 μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162 μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen. Copyright © 2016 Elsevier Ltd. All rights reserved.

Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rocchi, P.; Ferreri, A.M.; Capucci, A.

1981-01-01

Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (less than 8 x 10(-6)). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTr) mutants up to 1000-fold. The maximum recovery of DTr mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages.

Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rocchi, P.; Ferreri, A.M.; Capucci, A.

1981-01-01

Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (< 8 x 10/sup -6/). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTsup(r)) mutants up to 1000-fold. The maximum recovery of DTsup(r) mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages.

Plant toxicity, adaptive herbivory, and plant community dynamics

USGS Publications Warehouse

Feng, Z.; Liu, R.; DeAngelis, D.L.; Bryant, J.P.; Kielland, K.; Stuart, Chapin F.; Swihart, R.K.

2009-01-01

We model effects of interspecific plant competition, herbivory, and a plant's toxic defenses against herbivores on vegetation dynamics. The model predicts that, when a generalist herbivore feeds in the absence of plant toxins, adaptive foraging generally increases the probability of coexistence of plant species populations, because the herbivore switches more of its effort to whichever plant species is more common and accessible. In contrast, toxin-determined selective herbivory can drive plant succession toward dominance by the more toxic species, as previously documented in boreal forests and prairies. When the toxin concentrations in different plant species are similar, but species have different toxins with nonadditive effects, herbivores tend to diversify foraging efforts to avoid high intakes of any one toxin. This diversification leads the herbivore to focus more feeding on the less common plant species. Thus, uncommon plants may experience depensatory mortality from herbivory, reducing local species diversity. The depensatory effect of herbivory may inhibit the invasion of other plant species that are more palatable or have different toxins. These predictions were tested and confirmed in the Alaskan boreal forest. ?? 2009 Springer Science+Business Media, LLC.

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

PubMed

Fuchs, Evelyn; Haas, Alexander K; Spooner, Robert A; Yoshimura, Shin-ichiro; Lord, J Michael; Barr, Francis A

2007-06-18

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

PubMed

Rosskopf, Ute; Noeske, Kerstin; Werner, Esther

2005-01-01

The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.