Design, expression, and testing of a bivalent subunit botulism vaccine for use in cattle
Shtevi, Abraham (2009) Design, expression, and testing of a bivalent subunit botulism vaccine for use in cattle. PhD thesis, James Cook University.
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Botulism intoxication in cattle is a serious problem that needs to be addressed. Current vaccines offer protection but are based on lysed cells and inactivated toxin. These formulations are dangerous to produce and vary widely between batches. A subunit vaccine based on non-toxic components of the botulinum neurotoxin would lead to safer vaccines, less in vivo testing and reduced costs.
The aims of the study were threefold. Genes encoding the non-toxic subunits of Clostridium botulinum serotypes C and D neurotoxins were to be redesigned and synthesized de novo to optimize expression in the methylotrophic yeast Pichia pastoris. The synthetic genes were to be transformed and expressed in the yeast and conditions determined for effective expression of both subunits. Finally the expressed proteins were to be tested in vivo in BALB/c mice to validate the completed protein expression work. Ultimately, the aim was to create a system that expressed high levels of both subunits C and D that were shown to be effective in vivo. This would then be transferred to industry and would lead to the commercialization of a new generation bivalent vaccine against botulism to be used on cattle.
The genetic work carried out yielded two genes that were codon optimized for expression in P. pastoris, had a higher GC content than the original sequence and whose protein products would not be glycosylated during secretion, owing to the targeted substitution of histidine codons for asparagines codons. The GC content was increased by 13% in the D subunit and by nearly 20% in the C subunit.
Yeast transformation and expression were carried out and led to the isolation of positive clones expressing the synthetic genes. Protein was isolated from both the cell pellet and the medium, indicating that the product was being secreted from the cells. Yields varied and reached nearly 14.5 mg/L for BoNT/C and more than 3 mg/L in the case of BoNT/D.
The in vivo trials tested the immunizing properties of the two expressed subunits. A number of conditions and parameters were tested in the mice and it was determined that the aluminium hydroxide (Al(OH)3) adjuvant was the most effective adjuvant in terms of eliciting an immune response and leading to immunity in mice. Up to 60% of the mice immunized with subunit C were protected against a challenge of up to 25 LD50 but more than that proved fatal to naïve and immunized mice alike. ELISA and Western hybridizations indicated that antibodies were raised in the mice, especially when the aluminium hydroxide adjuvant was included in the formulation.
|Item Type:||Thesis (PhD)|
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|Keywords:||botulism intoxication, cattle, botulism vaccines, neurotoxins, subunit vaccines, vaccine production, gene synthesis, bivalent vaccines, biotechnology, vaccine expression|
|FoR Codes:||07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070710 Veterinary Pharmacology @ 100%|
|SEO Codes:||97 EXPANDING KNOWLEDGE > 970107 Expanding Knowledge in the Agricultural and Veterinary Sciences @ 100%|
|Deposited On:||25 Jun 2010 11:22|
|Last Modified:||12 Feb 2011 03:49|
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