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

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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 (HC) 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 HC, with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of HC, 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.

Introduction

Botulinum neurotoxin (BoNT) is secreted by the spore-forming bacterium Clostridium botulinum and is the most poisonous substance known.1 The crystal structure of BoNT serotype A (BoNT/A) shows three functional domains comprising a heavy and a light chain.2., 3., 4. The C-terminal portion of the heavy chain (HC) is the binding domain that docks the toxin to sialoganglioside receptors and a protein receptor on presynaptic neurons, resulting in toxin endocytosis.5., 6., 7. The translocation domain (HN), at the N-terminal portion of the heavy chain, mediates escape of the toxin light chain (LC) from the endosome. Depending on serotype, the LC cleaves one or more members of the SNARE complex of proteins, blocking acetylcholine release.8., 9.

Human botulism is caused by BoNT serotypes A, B, E, and F, and is characterized by flaccid paralysis which, if not fatal, requires prolonged hospitalization in an intensive care unit and mechanical ventilation. Naturally occurring botulism results from ingestion of contaminated food, anaerobic wound infections, or gastrointestinal tract colonization by clostridial bacteria†.10 Botulinum neurotoxins are classified by the Centers for Disease Control (CDC) as one of the six highest-risk threat agents for bioterrorism (the Class A agents) due to their extreme potency and lethality.11 Both Iraq and the former Soviet Union produced BoNT for use as weapons,12., 13. and the Japanese cult Aum Shinrikyo attempted to use BoNT for bioterrorism.11 Consequently, specific pharmaceutical agents are needed for treatment of intoxication.

Treatment of botulism in adults relies on the use of an antitoxin,14 currently generated from immunized horses.15 Unfortunately, this product is associated with a high incidence of side-effects, including serum sickness and anaphylactic shock. As an alternative, monoclonal antibody (mAb)-based antitoxins are under development.16., 17. Nowakowski and co-workers reported the generation of three mAbs, S25, C25, and 3D12, that neutralized BoNT/A both in vitro and in vivo.17 While in vivo neutralization for single mAbs was of relatively low potency, combining any two or all three mAbs led to highly potent neutralization of BoNT/A. Higher-affinity derivatives of these three mAbs are now in cGMP production for anticipated toxicology studies and human clinical trials‡.

We have been interested in mapping the epitopes of these and other BoNT/A mAbs. Such mapping can lead to an improved understanding of mechanism(s) of toxin neutralization, as well as shed light on the relationship between toxin structure and function. For example, putative sialoganglioside binding sites on the toxin have been identified using X-ray crystallography.18., 19. Are these sites where neutralizing mAbs bind? Similarly, the BoNT/A docking site for the protein receptor is unknown; identification of the epitopes for potently neutralizing BoNT/A mAbs might identify potential protein-receptor binding sites on the BoNT/A HC. Finally, marked synergy in toxin neutralization has been observed when mAbs are combined.17 Identifying the sites and interactions between toxin and neutralizing mAbs could provide a structural model of the immune complexes and perhaps provide insights into the mechanism of neutralization.

For this work, yeast display20., 21. was utilized to display all three BoNT/A domains, which could be used to map a panel of six mAbs to the domain level. This approach should prove useful for gross mapping of both polyclonal and monoclonal antibodies to BoNT/A, and can probably be adapted for domains of other BoNT/A subtypes or other BoNT serotypes. This approach has an advantage of not requiring native protein expression and purification. Using yeast-displayed libraries, the fine epitopes of neutralizing mAbs 3D12, HuC25, and S25 were identified down to the energetically important amino acid side-chains. The results identify a number of neutralizing epitopes on BoNT/A and suggest mechanisms of toxin neutralization.

Section snippets

Domain epitope mapping of BoNT/A-neutralizing antibodies

To display the three functional domains of botulinum neurotoxin type A (BoNT/A HC, HN, and LC) on the surface of yeast, we used PCR to amplify the relevant gene from plasmids containing either a synthetic HC gene, or a synthetic LC-HN gene (Figure 1). PCR fragments encoding HC, LC, HN, and LC-HN were then ligated into the yeast display vector pYD222 in-frame with an SV5 epitope tag. This allowed for C-terminal fusion of the SV5 tag and a means to measure the level of display on the yeast

Discussion

The three functional domains of BoNT/A were displayed on the surface of yeast, allowing the domain epitope mapping of a panel of six monoclonal antibodies. It was possible to display a pair of domains (HN–LC, 100 kDa), the largest protein displayed on the surface of yeast.21 At least three of these mAbs (HuC25, 3D12, and S25) are known to bind conformational epitopes on the HC.23 One of the BoNT/A mAbs studied (ING1) bound HN–LC, but did not bind HN or LC. A potential epitope for this mAb would

Strains, media and antibodies

The yeast strain EBY100Zeo was a gift from M. J. Feldhaus (Pacific Northwest National Laboratory, Richland, WA). Briefly, EBY100Zeo was derived from EBY100 (GAL1-AGA1:URA3 ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL) and carries the pTEF1 promoter zeocin-resistance gene (Sh ble gene). EBY100Zeo was maintained in YPD medium§. The E. coli strain DH5α, (K12, Δ(lac-pro), supE, thi, hsdD5/F traD36, proA+B+, lac

Acknowledgements

This work was supported, in part, by NIAID cooperative agreement U01 AI056493 and DoD contract DAMD17-03-C-0076.

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