Recombinant neurotoxin fragments of clostridium botulinum as protective antigens

Abstract

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are highly potent bacterial exotoxins responsible for the disease ``botulism``. They are composed of light chain (50 kDa) and heavy chain (100 kDa). In the present study, it was determined that various recombinant BoNT heavy chain fragments can be used for protective antigens. For this purpose, each C- and N-terminal region of BoNT/A and B (BoNT/B) heavy chain was expressed. Also, the C-terminal regions of BoNT/A and BoNT/B heavy chain genes and N-terminal regions of them were fused and expressed separately. These expressed proteins were purified by affinity chromatography and those were used to immunize female BALB/c mice. In order to determine whether the immunized mice were able to produce antibodies specific to the toxins, the sera from the immunized mice were analyzed by immunoblotting and ELISA. All of these recombinant proteins were able to elicit an antibody response to specific types of toxins. Furthermore, the antiserum from heavy chain N-terminal region of BoNT/A showed cross-reactivity with BoNT/B. Sixteen days after last immunization, the mice were challenged with 10-fold of $LD_50$ of BoNT/A and BoNT/B. All immunized mice were protected or partially protected from the death, which was caused by botulinum intoxication. These results suggested that some parts of the BoNT heavy chain C-terminal and N-terminal region elicit protective immune response, and therefore the vaccine for botulism include these fragments can be more effective.