title: Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.
abstract: 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-RVGPfusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVGPfusion protein was able to cross the in celluloBBB and neutralize RABV.
authors.affiliation: Institute for Infection and Immunity, St. George's Hospital Medical School, University of London, London, UK.|Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Waranyoo
authors.initials: W
authors.lastname: Phoolcharoen
authors.affiliation: Unité de Neuroimmunologie Virale, Département de Virologie, Institut Pasteur, Paris, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Prehaud
authors.affiliation: Institute for Infection and Immunity, St. George's Hospital Medical School, University of London, London, UK.
authors.forename: Craig J
authors.initials: CJ
authors.lastname: van Dolleweerd
authors.affiliation: Institute for Infection and Immunity, St. George's Hospital Medical School, University of London, London, UK.
authors.forename: Leonard
authors.initials: L
authors.lastname: Both
authors.affiliation: Unité de Neuroimmunologie Virale, Département de Virologie, Institut Pasteur, Paris, France.
authors.forename: Anaelle
authors.initials: A
authors.lastname: da Costa
authors.affiliation: Unité de Neuroimmunologie Virale, Département de Virologie, Institut Pasteur, Paris, France.
authors.forename: Monique
authors.initials: M
authors.lastname: Lafon
authors.affiliation: Institute for Infection and Immunity, St. George's Hospital Medical School, University of London, London, UK.
authors.forename: Julian K-C
authors.initials: JK
authors.lastname: Ma
mesh_terms: D057134:Antibodies, Neutralizing / Q000096:biosynthesis; D001812:Blood-Brain Barrier; D002460:Cell Line; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology*; D046549:Plantibodies / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D030821:Plants, Genetically Modified; D011820:Rabies virus / Q000276:immunology*; D011978:Receptors, Nicotinic / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014026:Nicotiana; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D006023:Glycoproteins; D010446:Peptide Fragments; D046549:Plantibodies; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C583075:rabies virus glycoprotein peptide
keywords: antibody engineering; blood-brain barrier; plant biotechnology; rabies virus; single-chain antibody