Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the<i>Clostridium botulinum</i>Neurotoxin Type A

Wu, Han-Chung; Yeh, Chia-Tsui; Huang, Yichen; Tarn, Lih-Jeng; Lung, Chien‐Cheng

doi:10.1128/aem.67.7.3201-3207.2001

Cited by 34 publications

(26 citation statements)

References 43 publications

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“…In the case of epsilon-toxin-mediated enterotoxemia, administration of toxin-neutralizing antiserum is used prophylactically to treat unvaccinated animals in the event of an outbreak of enterotoxemia (2). Epitopes recognized by antibodies that neutralize the activities of several select agent toxins have been mapped and provide valuable information that may be used in the development of novel antitoxin therapies (3,8,16,24,42,43,57,58).…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

McClain¹,

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2007

Infect Immun

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The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.The Clostridium perfringens species is divided into five types, A through E, based on production of the four "major toxins," the alpha-, beta-, epsilon-, and iota-toxins (50). Epsilon-toxin is one of the toxins produced by type B and D strains (44). The epsilon-toxin can lead to a fatal illness (enterotoxemia) in a variety of livestock animals, most frequently in sheep (50). Clinical signs in intoxicated sheep may include colic, diarrhea, and numerous neurological symptoms. Postmortem analysis reveals widespread increases in vascular permeability with cerebral, cardiac, pulmonary, and kidney edema (52, 53). Experimental intoxication of mice and rats with epsilon-toxin causes a rapidly fatal illness and pathological changes similar to those seen in livestock (12,13,28,46). The dose of epsilon-toxin required to kill 50% of mice has been estimated at between 65 and 110 ng per kg (27), which indicates that epsilon-toxin is one of the most potent known bacterial protein toxins (14). Despite reports of epsilon-toxin-producing C. perfringens being isolated from humans, it is unclear whether or not epsilontoxin causes illness in humans (15,22,26,31,34,49). Due to the clear threat posed to livestock and the potential threat to human health, the epsilon-toxin is classified as a category B overlap select agent by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture.To protect livestock from epsilon-toxin, both a vaccine (based on formalin-inactivated epsilon-toxin) and an equinederived antitoxin are available. Due to the rapid progression of the disease among livestock animals, treatment is generally not possible or practical, and the emphasis is placed on prevention either by vaccination or by administration of ant...

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Section: Discussionmentioning

confidence: 99%

Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

McClain¹,

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2007

Infect Immun

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“…There are many reports that the linear B-cell epitopes were characterized as neutralizing antibodies as in Clostridium botulinum neurotoxin type A (Btx A)[24]. In Bcipep, there are 748 neutralizing anti-peptide antibodies entries.…”

Section: Potential Utility and Limitationsmentioning

confidence: 99%

Bcipep: A database of B-cell epitopes

2005

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“…An LD 50 of 10 pg per mouse was reported for BoNT=A when administered by inter-peritoneal injection, which allows a detection limit of 7-20 pg=mL. (8,10,11) While highly sensitive, the mouse bioassay requires skilled personnel, uses a large number of mice, and can take up to 4 days to complete. It is clear that a rapid and simple-to-use test for presence of botulism toxin is required.…”

Section: Introductionmentioning

confidence: 98%

Characterization of the Epitope Region of F1-2 and F1-5, Two Monoclonal Antibodies to Botulinum Neurotoxin Type A

2009

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F1-2 and F1-5 are mouse IgG1 monoclonal antibodies that bind the heavy chain of Botulinum neurotoxin serotype A (BoNT/A). To characterize the epitopes of F1-2 and F1-5, three complementary experimental approaches were selected. First, recombinant peptide fragments of BoNT/A heavy-chain were used in Western blots to identify the epitope regions. Second, a peptide phage display library was used to identify specific amino acids bound by F1-2 and F1-5, and these amino acids were mapped onto the three-dimensional structure of BoNT/A. Third, selected amino acids were mutated to alanine and the effects of the mutations on F1-2 and F1-5 binding were evaluated. Data from recombinant peptide fragment binding experiments suggested that the epitopes for antibodies F1-2 and F1-5 are located between amino acids R564 and S793 on the toxin heavy chain. Furthermore, elimination of amino acids from the amino terminus (R564-K595), or from the carboxyl terminus (N759-S793) of this fragment abolished binding of both F1-2 and F1-5, suggesting a conformational epitope for these antibodies. Peptide sequences deduced from antibody binding to the peptide phage display library suggested that tyrosine residues located at positions 748, 750, and 753 might form a significant part of the F1-2 and F1-5 epitope motif. Mutation of Y750 or Y753 to alanine significantly reduced binding of either antibody, while mutation of Y748 to alanine had no effect on antibody binding. The nucleotide and deduced amino acid sequences of the variable regions of the heavy chains of F1-2 and F1-5 are reported. The complementarity determining regions (CDRs) of the heavy chains were found to be 78% identical. It is possible that F1-2 and F1-5 bind the same epitope via the common amino acids within their CDRs.

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Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on theClostridium botulinumNeurotoxin Type A

Cited by 34 publications

References 43 publications

Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

Bcipep: A database of B-cell epitopes

Characterization of the Epitope Region of F1-2 and F1-5, Two Monoclonal Antibodies to Botulinum Neurotoxin Type A

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