Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A

Liu, Fujia; Shi, Danyang; Mao, Yunyun; Xiong, Xiang-Hua; Lu, Jian-Sheng; Pang, Xiaobin; Dong, Xiaojie; Yang, Zhengming; Yu, Yun-Zhou

doi:10.1016/j.vaccine.2020.02.060

Cited by 10 publications

(11 citation statements)

References 26 publications

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“…Protection against BoNTs was achieved using a bivalent recombinant vaccine for BoNT/A and B based on their translocation and effector domains [35]. Recently, we explored the immunological characterization and immunoprotective efficacy of BoNT/A functional fragment antigens [36]. Our results showed that a recombinant E.coli-expressed AL-HN protein without formaldehyde treatment displayed a stable structure and antigenicity.…”

Section: Discussionmentioning

confidence: 96%

“…To produce recombinant functional fragment antigens of BoNT/E in Escherichia coli, gene fragments of each function fragment (EL, EHN, EL-HN, EHc, EHc-N, and EHc-C, Table 1) were ligated into a prokaryotic expression vector, pTIG-Trx, to produce recombinant expression plasmids. As previously reported [26,36], our laboratory constructed pTIG-Trx prokaryotic expression plasmids encoding the following BoNT/E molecule fragments with His-tag: EL (residues 1-422), EHN (residues 423-840), EL-HN (residues 1-840), EHc-N (residues 841-1063), EHc-C (residues 1056-1252), and EHc (residues 841-1252). The resulting recombinant plasmids were confirmed by sequencing.…”

Section: Production Of Recombinant Functional Fragment Antigens Deriv...mentioning

confidence: 99%

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Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E

Lu²,

Tan

et al. 2022

Toxins

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Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most toxic known protein and the causative agent of human botulism. BoNTs have similar structures and functions, comprising three functional domains: catalytic domain (L), translocation domain (HN), and receptor-binding domain (Hc). In the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. The EL-HN fragment (L and HN domains of BoNT/E) retained the enzymatic activity without in vivo neurotoxicity. Extensive investigations showed EL-HN functional fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Further experiments demonstrated the EL-HN provided a superior protective effect compared with the EHc or EHc and EL-HN combination. Thus, the EL-HN played an important role in immune protection against BoNT/E and could provide an excellent platform for the design of botulinum vaccines and neutralizing antibodies. The EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine.

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Section: Discussionmentioning

confidence: 96%

Section: Production Of Recombinant Functional Fragment Antigens Deriv...mentioning

confidence: 99%

Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E

Lu²,

Tan

et al. 2022

Toxins

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“…Extensive research have shown that the Hc domain is the main target of BoNT recombinant subunit vaccines, and has protective antigenic properties [ 4 , 25 , 26 , 27 , 28 , 29 ]. Currently, the recombinant Hc subunit vaccines of BoNT/A, B, E, and F all have good protection against the biologically active BoNTs of their own serotype after immunization [ 30 , 31 , 32 , 33 , 34 ]. The L and HN domains of BoNTs have also been developed as recombinant subunit vaccines with strong potency to prevent animal botulism [ 30 , 35 , 36 , 37 , 38 ].…”

Section: Introductionmentioning

confidence: 99%

“…Currently, the recombinant Hc subunit vaccines of BoNT/A, B, E, and F all have good protection against the biologically active BoNTs of their own serotype after immunization [ 30 , 31 , 32 , 33 , 34 ]. The L and HN domains of BoNTs have also been developed as recombinant subunit vaccines with strong potency to prevent animal botulism [ 30 , 35 , 36 , 37 , 38 ].…”

Section: Introductionmentioning

confidence: 99%

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Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F

Lu²,

Liu

et al. 2023

Toxins

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Botulinum neurotoxins (BoNTs) can cause nerve paralysis syndrome in mammals and other vertebrates. BoNTs are the most toxic biotoxins known and are classified as Class A biological warfare agents. BoNTs are mainly divided into seven serotypes A-G and new neurotoxins BoNT/H and BoNT/X, which have similar functions. BoNT proteins are 150 kDa polypeptide consisting of two chains and three domains: the light chain (L, catalytic domain, 50 kDa) and the heavy chain (H, 100 kDa), which can be divided into an N-terminal membrane translocation domain (HN, 50 kDa) and a C-terminal receptor binding domain (Hc, 50 kDa). In current study, we explored the immunoprotective efficacy of each functional molecule of BoNT/F and the biological characteristics of the light chain-heavy N-terminal domain (FL-HN). The two structure forms of FL-HN (i.e., FL-HN-SC: single chain FL-HN and FL-HN-DC: di-chain FL-HN) were developed and identified. FL-HN-SC could cleave the vesicle associated membrane protein 2 (VAMP2) substrate protein in vitro as FL-HN-DC or FL. While only FL-HN-DC had neurotoxicity and could enter neuro-2a cells to cleave VAMP2. Our results showed that the FL-HN-SC had a better immune protection effect than the Hc of BoNT/F (FHc), which indicated that L-HN-SC, as an antigen, provided the strongest protective effects against BoNT/F among all the tested functional molecules. Further in-depth research on the different molecular forms of FL-HN suggested that there were some important antibody epitopes at the L-HN junction of BoNT/F. Thus, FL-HN-SC could be used as a subunit vaccine to replace the FHc subunit vaccine and/or toxoid vaccine, and to develop antibody immune molecules targeting L and HN domains rather than the FHc domain. FL-HN-DC could be used as a new functional molecule to evaluate and explore the structure and activity of toxin molecules. Further exploration of the biological activity and molecular mechanism of the functional FL-HN or BoNT/F is warranted.

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Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs

Shi¹,

Lu²,

Mao³

et al. 2023

Appl Microbiol Biotechnol

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Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab′) 2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins. Key points • Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made. • Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain. • A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12515-2.

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Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A

Cited by 10 publications

References 26 publications

Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E

Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E

Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F

Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs

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