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Abstract
Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')2, Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination.
Highlights
Seven serologically distinct botulinum toxins, BoNT/A to/G, are produced by different strains of the Gram-positive, sporeforming anaerobic bacterium Clostridium botulinum
In order to compare the protective activity of TA12 monoclonal anti-BoNT/A antibodies (mAbs) with other recent reported mAbs, we studied the ability of a fixed amount of TA12 antibody (50 mg/mouse) to protect mice challenged with increasing amount of BoNT/A1 ranging from 100–10,000 MLD50 (Figure 1A)
12 out of 14 mAbs presented neutralizing activity and recognized 4 different epitopes of the toxin-binding domain, which was used as antigen
Summary
Seven serologically distinct botulinum toxins, BoNT/A to/G, are produced by different strains of the Gram-positive, sporeforming anaerobic bacterium Clostridium botulinum. Since no drugs allow prevention or treatment, toxin-neutralizing antibodies were developed for prophylactic or therapeutic treatment [3] These antitoxins were obtained after immunization of several species: horse [4], goat [5], mouse [6,7], and human [8]. Different strategies have been developed to circumvent these limitations using phage display libraries from immunized mice or humans [10,11] or using immunoglobulin fragments, like F(ab’)2 [12,13], which are less immunogenic. These fragments have short in vivo halflives, which must be taken into account considering the expected duration of antitoxin activity. Several reports have shown that linking polyethylene glycol (PEG) molecules to F(ab’) fragments (pegylated fragments) can overcome this problem by extending in vivo half-life [14,15,16,17]
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