Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non‐human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as ‘category A’ pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost‐effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C‐terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co‐expression of the GP1‐heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size‐exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti‐Ebola virus antibody production at levels comparable to those obtained with a GP1 virus‐like particle. These results show excellent potential for a plant‐expressed EIC as a human vaccine.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.     

Rapid transient expression of cholera toxin B subunit (CTB) in Nicotiana benthamiana

Cholera toxin B subunit (CTB) has the potential to be an effective adjuvant for mucosal vaccines because of its ability to increase antigen uptake and presentation by antigen-presenting cells through GM1-ganglioside binding. CTB has been produced using different recombinant protein expression systems. This study used the geminiviral replicon system to transiently express CTB in Nicotiana benthamiana. The plant-optimized CTB gene was cloned into a geminiviral vector and infiltrated into N. benthamiana leaves. The highest CTB protein level was observed on day 4 with approximately 4 μg/g fresh weight. The Western blot analysis using anti-CTB suggests assembly of CTB into oligomers. Based on the GM1-ELISA results, this CTB transiently expressed in plants showed biological activity for binding the intestinal epithelial cell membrane glycolipid receptor, GM1-glanglioside, which implies its potential as an adjuvant for mucosal vaccines.