Production of monoclonal antibodies against botulinum neurotoxin in Nicotiana benthamiana.

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.

Plant-Produced S1 Subunit Protein of SARS-CoV-2 Elicits Immunogenic Responses in Mice.

SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic. The virus spreads rapidly with a high transmission rate among humans, and hence virus management has been challenging owing to finding specific therapies or vaccinations. Hence, an effective, low-cost vaccine is urgently required. In this study, the immunogenicity of the plant-produced S1 subunit protein of SARS-CoV-2 was examined in order to assess it as a potential candidate for SARS-CoV-2. The SARS-CoV-2 S1-Fc fusion protein was transiently produced in Nicotiana benthamiana. Within four days of infiltration, the SARS-CoV-2 S1-Fc protein was expressed in high quantities, and using protein A affinity column chromatography, plant-produced S1-Fc protein was purified from the crude extracts. The characterization of plant-produced S1-Fc protein was analyzed by SDS-PAGE and Western blotting. Immunogenicity of the purified S1-Fc protein formulated with alum induced both RBD specific antibodies and T cell immune responses in mice. These preliminary results indicated that the plant-produced S1 protein is immunogenic in mice.