Production of monoclonal antibodies against botulinum neurotoxin in Nicotiana benthamiana

ABSTRACT

Botulism is a fatal neurologic disease caused by the botulinum toxin (BoNT) produced by Clostridium botulinum. It is a rare but highly toxic disease with symptoms, such as cramps, nausea, vomiting, diarrhea, dysphagia, respiratory failure, muscle weakness, and even death. Currently, two types of antitoxin are used: equine-derived heptavalent antitoxin and human-derived immunoglobulin (BabyBIG®). However, heptavalent treatment may result in hypersensitivity, whereas BabyBIG®, has a low yield. The present study focused on the development of three anti-BoNT monoclonal antibodies (mAbs), 1B18, C25, and M2, in Nicotiana benthamiana. The plant-expressed mAbs were purified and examined for size, purity and integrity by SDS-PAGE, western blotting and size-exclusion chromatography. Analysis showed that plant-produced anti-BoNT mAbs can fully assemble in plants, can be purified in a single purification step, and mostly remain as monomeric proteins. The efficiency of anti-BoNT mAbs binding to BoNT/A and B was then tested. Plant-produced 1B18 retained its ability to recognize both mBoNT/A1 and ciBoNT/B1. At the same time, the binding specificities of two other mAbs were determined: C25 for mBoNT/A1 and M2 for ciBoNT/B1. In conclusion, our results confirm the use of plants as an alternative platform for the production of anti-BoNT mAbs. This plant-based technology will serve as a versatile system for the development botulism immunotherapeutics.

KEYWORDS:

• Botulism
• botulinum neurotoxin
• Clostridium botulinum
• monoclonal antibody
• Nicotiana benthamiana

Disclosure statement

Waranyoo Phoolcharoen is a founder/shareholder of Baiya Phytopharm Co., Ltd. Christine Joy I. Bulaon, Kaewta Rattanapisit, and Apidsada Wongwatanasin are employees of Baiya Phytopharm Co., Ltd. No potential conflict of interest was reported by the remaining authors.

Author contributions statement

Waranyoo Phoolcharoen conceived and designed the study. Kornchanok Sangprasat, Christine Joy I. Bulaon, Kaewta Rattanapisit, and Perawat Jirarojwattana performed construct design for gene synthesis. Kornchanok Sangprasat and Apidsada Wongwatanasin performed gene cloning. Kornchanok Sangprasat performed expression, purification experiments, SDS-PAGE, western blot analysis. Kornchanok Sangprasat and Theerakarn Srisangsung performed in vitro binding assay. Kornchanok Sangprasat interpreted the data. Kornchanok Sangprasat and Christine Joy I. Bulaon drafted the manuscript. All authors reviewed the manuscript.

Data availability statement

The data presented in this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This study was supported by the National Vaccine Institute (NVI) of Thailand Science Research and Innovation, the Second Century Fund (C2F) scholarship of Chulalongkorn University, Thailand, the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research, and Innovation [grant number B13F660137], and the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Fund.