Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display.

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.

Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization.

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.

A designed four helix bundle protein with native-like structure.

A 108 amino acid protein was designed and constructed from a reduced alphabet of seven amino acids. The 2.9 A resolution X-ray crystal structure confirms that the protein is a four helix bundle, as it was designed to be. Hydrogen/deuterium exchange experiments reveal buried amide protons with protection factors in excess of 1 x 10(6) in the range characteristic of well protected protons in functional folded proteins (10(3)-10(8)) rather than protons in rapid exchange (0-10(2)). The protein is monomeric at 1 mM, the concentration at which the exchange experiments were undertaken, indicating that the exchange factors are due to a unique stable tertiary structure fold, and not due to any higher order quaternary structure. Thermodynamic analysis provides an estimate of the free energy of folding of -9.3 kcal mole-1 at 25 degrees C, consistent with the free energy of folding derived from the protection factors of the most protected protons, indicating that global unfolding is required for exchange of the most protected protons.

The application of malononitriles as microviscosity probes in pharmaceutical systems.

Three molecules were investigated for their ability to distinguish variations in the microviscosity of the surrounding medium. Julolidinemalononitrile (JMN), p-(N-dimethylaminobenzylidene) malononitrile (BMN), and p-(N-dimethylaminocinnamylidene) malononitrile (CMN) were dissolved in media of various micro- and bulk viscosities. The fluorescence intensity of each dissolved probe and the bulk viscosity of each medium were measured. In solutions of low molecular weight substances, where the micro- and bulk viscosities are expected to correspond, the fluorescence behavior of each probe was a function of bulk viscosity and was independent of solution composition. In contrast, in aqueous solutions of methylcellulose, the fluorescence behavior of the probes corresponds to microviscosities significantly lower than the measured bulk viscosities. Thus, the probes are useful in resolving the microviscosity from bulk viscosity of neat liquid and solution systems. The sensitivity of the probes to viscosity is in the order JMN > BMN > CMN. Due to its limited water solubility, JMN is not particularly useful for pharmaceutical systems. CMN is the preferred probe for these applications due to its high fluorescence intensity over a large viscosity range.