Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization

Abstract

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.

FIG. 1.

Analysis of BoNT/A gene sequences. (A) Phylogenetic tree of BoNT/A genes reveals two clusters, A1 and A2. (B) Homology of the different domains of BoNT/A1 and BoNT/A2. (C) Model of the amino acid side chain differences between BoNT/A1 and BoNT/A2. Side-chain differences between BoNT/A2 and BoNT/A1 were modeled into the X-ray crystal structure of BoNT/A1 (3BTA [31]) using Modeler (43) in the Insight suite (Accelrys, San Diego, CA). Two different views of the toxin are shown rotated 180° about the y axis (Pymol; Delano Scientific, South San Francisco, CA). The BoNT/A heavy-chain binding domain is in white at the top of the figure, with the putative ganglioside binding residues (3BTA E1202, H1252, and W1265) in blue with the ganglioside (red) modeled by superposition using the O suite program LSQMAN (24) from the BoNT/B crystal structure (1F31 [49]). The root mean square deviation between the three ganglioside binding residue C-α positions from BotNT/A and BotNT/B is 0.22 Å. These residues are in the C-terminal subdomain, for which the root mean square deviation between the binding domain structures (3BTA residues 1091 to 1295 and 1F31 residues 1092 to 1290) is 1.39 Å. The heavy-chain translocation domain is in orange and the light-chain catalytic domain is in white at the bottom of the figure. Side-chain differences between BoNT/A1 and BoNT/A2 toxins are shown in green.

FIG. 2.

Analysis of BoNT/B gene sequences. Phylogenetic tree of BoNT/B genes reveals four clusters: BoNT/B1, BoNT/B2, nonproteolytic BoNT/B, and bivalent BoNT/B. Percent differences between clusters range from 3.6 to 7.7%. As with BoNT/A, the greatest differences are seen in the heavy chain.

FIG. 3.

Binding of BoNT/A monoclonal antibodies to BoNT/A1 and BoNT/A2 toxins as determined by capture ELISA. Wells were coated with the indicated MAb followed by various concentrations of pure or complex BoNT/A1 or BoNT/A2. Toxin binding was detected using polyclonal equine BoNT/A antisera. A1 toxins are indicated by solid squares; A2 toxins are indicated by open circles. Pure toxins are solid lines; toxin complexes are dashed lines. Differences in binding signals for MAbs C25 and B4 between A1 and A2 pure and complex toxins may reflect the presence of different sets of accessory proteins (reference and see the text).

FIG. 4.

Ability of MAb pairs to protect mice challenged with BoNT/A1 toxin. A range of mouse LD₅₀s of BoNT/A1 toxin complex was mixed with 50 μg of an equimolar ratio of the indicated MAbs, and the mixture was injected intraperitoneally. The number of mice surviving versus challenge dose is indicated.

FIG. 5.

Ability of MAb triplets to protect mice challenged with BoNT/A1 or BoNT/A2 toxins. A range of mouse LD₅₀s of BoNT/A1 toxin complex (A) or BoNT/A2 toxin complex (B) was mixed with 50 μg of an equimolar ratio of the indicated MAbs, and the mixture was injected intraperitoneally. The number of mice surviving versus challenge dose is indicated.