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.

Mucosal Vaccine Targeting Improves Onset of Mucosal and Systemic Immunity to Botulinum Neurotoxin A

“…This additional level of protection is far superior to that of the conventional pentavalent BoNT vaccine not optimized to include mucosal anti-BoNT immunity. A final disadvantage is that, although traditionally BoNTs have been grouped as one of seven serotypes, it has become apparent that each serotype is actually composed of a family of closely related subtypes in which the primary sequence of each BoNT/A subtype can vary by as much as 32% (30). Therefore, it would be advantageous to produce a vaccine that can preserve as many of the neutralizing and cross-neutralizing epitopes among serotypes and serotype variants.…”

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

“…However, botulinum neurotoxins are known to be diverse [31,32], with multiple subtypes that present distinct challenges in designing effective prophylactic and therapeutic molecules. While some toxin subtypes differ by as much as 32%, only the C and D serotypes show specific mosaic patterns, which make protection after vaccination particularly challenging.…”

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

“…Human botulism is generally associated with exposures to BoNTs of the serotypes A, B, E, and F. In addition to distinct serotypes, many BoNT subtypes have been recently identified based on their sequence variations and antigenic differences. [4][5][6] For example, gene sequence analysis has identified five BoNT/A subtypes (A1 through A5). [7][8][9] Whereas the sequence similarity among different serotypes is relatively low, the subtypes within a BoNT serotype generally exhibit high sequence similarity.…”