C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 μg), Hn (5 μg), Hc (5 μg), or a mixture of Hn (5 μg) and Hc (5 μg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10⁶ the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10⁵ to 10⁶ MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 μg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused- or GST eliminated-preparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 μg, is useful for vaccine trials in mice.