Purification and Characterization of a Novel Subtype A3 Botulinum Neurotoxin

Abstract

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 ؋ 10 7 50% lethal doses (LD 50 )/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins.

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

“…with BoNT/A3 at 10 3-4 U/ml exhibited botulism intoxication symptoms that were uniquely different from those of mice injected with A1 at equal concentrations (23). To determine if the other BoNT/A subtypes provoked unique intoxication symptoms, mice were injected i.v.…”

“…The 150-kDa holotoxins of BoNT/A1, -2, -3, and -5 were purified from C. botulinum strains Hall A hyper, Kyoto-F, CDC A3, and A661222 as previously described (23,(32)(33)(34). The purified toxins were stored in 40% glycerol-phosphate-buffered saline at Ϫ20°C until use.…”

“…Surviving and dead mice were counted and specific activities calculated using the method of Reed and Muench (38). 3 U of BoNT/A1, -2, or -5 in 100 l of gelatin phosphate buffer (32), as has previously been described for BoNT/A3 (23). Three mice per subtype were injected and injections repeated as necessary.…”

“…For BoNT/A3, intoxication symptoms significantly different from those seen with A1 were observed when it was injected intravenously (i.v.) in mice at high concentrations (10 3 U) and manifested first in paralysis of the front legs, hind legs, and whole body followed by death (23). In contrast, the typical symptoms of BoNT/A1 intoxication include ruffled fur, labored breathing, descending flaccid paralysis, and spasticity just prior to death (24).…”

“…Similar experiments investigating symptoms of intoxication have not been conducted for other subtypes. Additionally, various investigators have shown differences in the ability of monoclonal and polyclonal antibodies to bind to or neutralize various subtypes within the same serotype (23,(25)(26)(27).…”