CVI Accepts, published online ahead of print on 8 October 2008

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Clin. Vaccine Immunol. doi:10.1128/CVI.00239-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Expression of a recombinant Hc of Clostridium botulinum neurotoxin serotype F and its evaluation as an effective subunit vaccine

Yun-Zhou Yu, Na Li, Rui-Lin Wang, Heng-Qi Zhu, Shuang Wang, Wei-Yuan Yu, and Zhi-Wei Sun^*

Beijing Institute of Biotechnology, 20 Dong Dajie Street, Fengtai District, Beijing 100071, China

^* To whom correspondence should be addressed.

A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of Clostridium botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). Following the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 µg FHc were completely protected against an intraperitoneal administration of 20, 000 50% lethal doses (LD₅₀) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using alhydrogel adjuvant via the intramuscular route and subsequently challenged with 20, 000 LD₅₀ of neurotoxin serotype F. A dose-response was observed in both the antibody titer and protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 µg or two injections 0.04 µg FHc were completely protected. These findings suggest that the recombinant FHc expressed in Escherichia coli is efficacious in protecting mice against challenge with active botulinum neurotoxin serotype F and the recombinant FHc subunit vaccine may be useful in humans.