Full text

Turn on search term navigation

Copyright: © 2025 Quynh Pham N et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

Background

Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (H C) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively H C of other BoNT/A subtypes. This study aimed to generate H C domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains.

Methods

H C domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/H CA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for H C domains of BoNT/A2-A8 were derived from pET45b-H CA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of H C domains and to evaluate the reactivity of VHHs to these domains.

Results

SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the H C domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for H C of BoNT/A1, two new VHHs were found to recognize the H C domains of all BoNT/A subtypes, of which VHH-A3 displayed EC 50 values for these domains close to those of ciA-C2.

Conclusion

This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A.

Details

Title
Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity
Author
Quynh Pham Nga 1 ; Mai Tam Trang 2 ; Dang Tran Bao Anh 2 ; Huong Tran Ly 1 ; Mai, Vu Quynh 1 ; Trong Nguyen Chien 1 ; Thi Phuong Tran Anh 1 ; Dang Tran Nhat Minh 3 ; Tran Van Anh 3 ; Huy Tran Thinh 4 ; Tran Van Khanh 4 ; Quang Le Hoa 1   VIAFID ORCID Logo 

 School of Chemistry and Life Sciences, Hanoi University of Science and Technology, Hanoi, Vietnam 
 High School for the Gifted in Natural Sciences, Vietnam National University, Hanoi, 120558, Vietnam 
 Hanoi Medical University, Hanoi, 11521, Vietnam 
 Center for Gene and Protein Research, Hanoi Medical University, Hanoi, 11521, Vietnam 
University/institution
U.S. National Institutes of Health/National Library of Medicine
Publication year
2025
Publication date
2025
Publisher
Faculty of 1000 Ltd.
e-ISSN
20461402
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
3204257692
Copyright
Copyright: © 2025 Quynh Pham N et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.