Article

Fine and Domain-level Epitope Mapping of Botulinum Neurotoxin Type A Neutralizing Antibodies by Yeast Surface Display

Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, San Francisco General Hospital, CA 94110, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 02/2007; 365(1):196-210. DOI: 10.1016/j.jmb.2006.09.084
Source: PubMed

ABSTRACT

Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.

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    • "Supplemental Table 1 in the supplementary material lists the antibodies tested in developing the microarray. Antibodies were purchased from Gallus Immunotech (Cary, NC, USA), R&D Systems (Minneapolis, MN, USA), List Biological Laboratories (Campbell, CA, USA), Metabiologics, Abcam (Cambridge, MA, USA), Meridian Life Science (Memphis, TN, USA), and HyTest (Turku, Finland) or were obtained using yeast display technologies for molecular evolution and selection of antibodies in the laboratory of James D. Marks at the University of California, San Francisco [36] [37] [38] [39] [40]. To produce the high-affinity antibodies, human immunoglobulin G (IgG) monoclonal antibodies (mAbs) were first isolated from a single-chain variable fragment (scFv) library displayed on yeast that was generated from either a human volunteer or mice immunized with pentavalent BoNT toxoid. "
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    ABSTRACT: Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.
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    • "This function domain is essential for the binding to anionic lipid in the environment of lipid raft, which is important for the translocation processing of the toxin. [25], [26]. Interestingly, the neutralizing potency of the pair of mAbs (C25+3D12), which bind to translocation domain and sialoganglioside binding domain respectively, is 10 times higher than that of S25+3D12, whose epitopes are located at the same function domain. "
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    ABSTRACT: Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
    Preview · Article · Aug 2012 · PLoS ONE
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    • "Supplemental Table 1 in the supplementary material lists the antibodies tested in developing the microarray. Antibodies were purchased from Gallus Immunotech (Cary, NC, USA), R&D Systems (Minneapolis, MN, USA), List Biological Laboratories (Campbell, CA, USA), Metabiologics, Abcam (Cambridge, MA, USA), Meridian Life Science (Memphis, TN, USA), and HyTest (Turku, Finland) or were obtained using yeast display technologies for molecular evolution and selection of antibodies in the laboratory of James D. Marks at the University of California, San Francisco [36] [37] [38] [39] [40]. To produce the high-affinity antibodies, human immunoglobulin G (IgG) monoclonal antibodies (mAbs) were first isolated from a single-chain variable fragment (scFv) library displayed on yeast that was generated from either a human volunteer or mice immunized with pentavalent BoNT toxoid. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3fM (0.2pg/ml) to 14.7fM (2.2pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples.
    Full-text · Article · Aug 2012 · Analytical Biochemistry
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