Recombinant tetanus vaccine (Beijing Institute of Biotechnology/Sichuan ZehaTimes Pharmaceutical)

Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine.

In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems.

The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice.

The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity.

The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

Tetanus is an acute, fatal disease caused by exotoxin produced by Clostridium tetani. The current vaccine against tetanus is based on inactivated tetanus toxin (TeNT). To develop a recombinant TeNT vaccine suitable for replacement of full-length tetanus toxoid (TT) vaccine for use in humans, a recombinant non-tagged isoform of the Hc domain of the tetanus toxin (THc) was expressed in Escherichia coli and purified by sequential chromatography steps. The immunogenicity and protective effect of the THc antigen were explored and compared with those of TT in Balb/c mice. The THc-based subunit vaccine provided complete protection against TeNT challenge following a high dosage as a toxoid vaccine. While the anti-THc and neutralising antibody titres were higher for the THc-based vaccine than the TT vaccine because protective epitopes are located on the THc domain. Frequency- and dose-dependent immunoprotection were also observed in THc-immunised mice. Mice immunised with one injection of 1 μg or 4 μg THc antigen were completely protected against 10² or 10³ 50% mouse lethal dose (LD₅₀) of TeNT, respectively. Furthermore, the THc protein was found to recognise and bind to ganglioside GT1b in a dose-dependent manner, and anti-THc sera antibodies also inhibited binding between THc and GT1b. Antigen on the form of recombinant non-tagged THc domain expressed in E. coli achieved strong immunoprotective potency, suggesting that it could be developed into a candidate subunit vaccine against tetanus as an alternative to the current TT vaccine.