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Sequence Variation within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization

Toxinology Division, USAMRIID, Frederick, Maryland 21702,¹ Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, Room 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, California 94110,² Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin 53706³

Received 22 October 2004/ Returned for modification 25 January 2005/ Accepted 26 April 2005

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 (LD₅₀s) of A1 toxin but less than 500 LD₅₀s 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.

Editor: A. D. O'Brien

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