Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris

Dux, Michael P.; Huang, Jicai; Barent, Rick; İnan, Mehmet; Swanson, Scott R.; Sinha, Jai B. P.; Ross, John T.; Smith, Leonard A.; Smith, Theresa J.; Henderson, Ian; Meagher, Michael M.

doi:10.1016/j.pep.2010.09.016

Cited by 13 publications

(5 citation statements)

References 24 publications

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“…However, as most of the expressed protein was insoluble in this system, subsequent studies have used the alternative host Pichia pastoris. Using this system, H C fragments of botulinum A, B, C, D, E, and F have been produced with satisfactory yields (15)(16)(17)(18)(19)(20). It is important to note, however, that P. pastoris is considered to be a less attractive host than E. coli for recombinant gene expression, from the perspectives of both genetic manipulation and production processes, and there is still an interest in improv-from the vector pET-9a (T7 promoter) were grown overnight at 37°C without induction.…”

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confidence: 99%

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

David

Diamant

Barnea

et al. 2013

Clin Vaccine Immunol

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The receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (H C ), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed in Escherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg H C per liter). The product was highly immunogenic, protecting mice from a challenge with 10 5 50% lethal dose (LD 50 ) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with H C increased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first time in vivo inhibition of BoNT/A intoxication by H C /A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of H C /A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P ‫؍‬ 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of H C /A to cross-inhibit BoNT/E intoxication was evaluated. The administration of H C /A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P ‫؍‬ 0.003). Furthermore, a combination of H C /A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD 50 of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by H C /A.

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confidence: 99%

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

David

Diamant

Barnea

et al. 2013

Clin Vaccine Immunol

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“…This protein is supposed to function as a vaccine against botulism. Recombinant BoNT-Hc of other serotypes has also been produced (34,35), but serotype A is the most studied one among distinct known serotypes of BoNT, and since it causes human botulism, it was chosen as the serotype of interest in this research. Pichia pastoris is the selected organism to express the product since it has a high potential for expressing the protein of interest in large quantities.…”

Section: Discussionmentioning

confidence: 99%

“…There have been attempts to produce the recombinant BoNT/A-Hc in the GS115 strain of Pichia pastoris (25,26,35,39), but due to the advantages of the PichiaPink kit mentioned above, the expression of the recombinant product has been evaluated in the four strains of PichiaPink. Also, despite the failure of expressing the recombinant BoNT/A-Hc by X-33, this strain is proved to be efficient in expressing the BoNT/Hc of other serotypes such as C (34).…”

Section: Discussionmentioning

confidence: 99%

Expression, Purification, and Verification of Recombinant Botulinum Neurotoxin Type A Binding Domain: A Comparison Between X33 and PichiaPink Strains of Pichia pastoris

Ebrahimi

Abedi

2020

Jundishapur J Nat Pharm Prod

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Background: An effective method to develop a safe vaccine against botulism is to utilize molecular biology techniques to produce recombinant antigens, which provoke the immune response in the recipient organism. A suggested antigen is a specific recombinant fragment of the botulinum neurotoxin (BoNT), which elicits the predictable immune response and does not have any toxic effects. In this study, the binding domain of the heavy chain of BoNT serotype A, which is the responsible subunit for binding to the receptor(s) of presynaptic membranes in neuromuscular junctions, is the selected fragment of this toxin to be recombinantly produced. Objectives: In order to prevent a severe syndrome such as Botulism, developing efficient vaccines against it is a necessity. Efforts have been made to accomplish this throughout time; however, some have discontinued due to the risks and unreliability of their production and usage. Methods: The encoding gene of BoNT/A-Hc was cloned into two different strains of Pichia pastoris, which were compared to each other based on the yield of the recombinant product. Results: The results demonstrated that the expression of recombinant BoNT/A-Hc by PichiaPink strain was successful, and the achieved recombinant BoNT/A-Hc was subsequently purified and then verified by using the specific antibody and analytical methods. Conclusions: In contrast, the expression results from the X-33 strain were not significant.

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“…These studies revealed that antibodies developed both in humans and mice recognize only a few peptides out of the whole collection of overlapping synthetic peptides. For a long time, the epitope mapping efforts were mostly focused on the heavy chains of botulinum neurotoxins, as those chains were considered to be the most important for development of protective immunity against these toxins (Baldwin et al, 2008; Dux et al, 2011; Smith and Rusnak, 2007; Yu et al, 2009). Only recently were results of the first attempts to analyze the epitope composition of light chains published (Atassi et al, 2011; Atassi et al, 2012a; Atassi et al, 2012b).…”

Section: Introductionmentioning

confidence: 99%

Epitope mapping of botulinum neurotoxins light chains

Zdanovsky

Zdanovskaia

2012

Toxicon

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Botulinum neurotoxins (BoNTs) are listed among the most potent biothreat agents. Simultaneously, two out of seven known serotypes of these toxins are used in medicine and cosmetics. This situation calls for development of detailed epitope maps of these toxins. Such maps will help to develop new ways for decreasing damage caused by these toxins if they were to be used as weapons while retaining the therapeutic effect of these toxins used as medicine. Here, we used a library of random fragments of DNA encoding the catalytic domain of botulinum neurotoxin serotype A to identify short epitope-forming sequences. We demonstrated that knowledge of such sequences in a BoNT of one serotype can be used for identification of epitope-forming sequences in other serotypes of BoNTs. We also demonstrated a serodiagnostic value of identified sequences and their ability to retain epitope-specific structures and trigger production of corresponding antibodies, even when they are transferred into a background of a completely alien carrier protein.

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Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris

Cited by 13 publications

References 24 publications

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

Expression, Purification, and Verification of Recombinant Botulinum Neurotoxin Type A Binding Domain: A Comparison Between X33 and PichiaPink Strains of Pichia pastoris

Epitope mapping of botulinum neurotoxins light chains

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