Epitope Mapping of Neutralizing Botulinum Neurotoxin A Antibodies by Phage Display

Mullaney, Brian P.; Pallavicini, M. G.; Marks, James D.

doi:10.1128/iai.69.10.6511-6514.2001

Cited by 38 publications

(42 citation statements)

References 27 publications

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“…In fact, two spatially separated ganglioside binding sites have been observed in the co-crystal structure of the homologous tetanus toxin (32), and mAbs binding nonoverlapping tetanus toxin epitopes can block binding of toxin to GT1b ganglioside (33). Our prior epitope mapping studies are consistent with multiple mAbs blocking a large portion of the BoNT binding domain (H C ) (34). Two of the mAbs (S25 and 3D12) bind the C-terminal subdomain of BoNT H C .…”

Section: Figsupporting

confidence: 52%

“…The C25 mAb binds a conformational epitope that consists of sequence from the N-and C-terminal subdomains of BoNT H C . One model consistent with the epitope mapping places the three mAb epitopes on the same H C face and overlapping the known docking sites for the putative cellular ganglioside receptor GT1b (34). Validation of this model awaits finer epitope mapping studies.…”

Section: Figmentioning

confidence: 77%

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Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

Nowakowski

Wang

Powers

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT͞A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT͞A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. monoclonal antibody ͉ immunotherapy ͉ antibody engineering ͉ vaccine ͉ phage display

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Section: Figsupporting

confidence: 52%

Section: Figmentioning

confidence: 77%

Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

Nowakowski

Wang

Powers

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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305

321

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“…Four MAbs (3D12, C25, B4, and S25) bound to nonoverlapping epitopes on the BoNT/A H C , as determined by ELISA on recombinant H C . 3D12 and S25 have been previously epitope mapped to the C-terminal subdomain of BoNT/A H C , while C25 recognizes a complex epitope formed by the two H C subdomains (37). One MAb (9D8) bound the BoNT/A translocation domain (H N ) as determined by ELISA on recombinant H N (data not shown).…”

Section: Sequence Variation Within Botulinum Neurotoxin Serotypesmentioning

confidence: 99%

Sequence Variation within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization

et al. 2005

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The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500-to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD 50 s) of A1 toxin but less than 500 LD 50 s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.

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“…Since an analysis of only 48 published full-length toxin gene sequences revealed the presence of 18 different subtypes, it is likely that additional subtypes might exist (39). Defining the extent of such toxin diversity is a first step in the development of detection systems and countermeasures for the prevention and treatment of botulism (33,39). In addition, analysis of a large population of strains can be used to better understand the evolutionary relationship between the toxin moieties and the genomic backgrounds that contain these toxins.…”

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confidence: 99%

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

et al. 2007

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Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.Clostridium botulinum is a taxonomic collection of several distinct species of anaerobic gram-positive spore-forming bacteria that produce the most poisonous substance known, botulinum neurotoxin (BoNT) (1,8). These organisms, along with related neurotoxin-producing species that, for a variety of reasons, were not included under the C. botulinum taxon, pose global health problems that affect both infant and adult humans and can also affect wildlife, waterfowl, and domestic animals. They cause intoxication through ingestion of the neurotoxin in contaminated foods. Toxicoinfections can also occur after contact with bacteria or bacterial spores (6, 17). These pathogens are ubiquitous and can be found in soils and sedi-

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Epitope Mapping of Neutralizing Botulinum Neurotoxin A Antibodies by Phage Display

Cited by 38 publications

References 27 publications

Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

Sequence Variation within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

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