title: Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases.
abstract: Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K(D) near 1 nM) and include potent inhibitors of BoNT/A protease activity (K(i) near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.
authors.affiliation: Tufts Cummings School of Veterinary Medicine, Department of Biomedical Sciences, 200 Westboro Road, North Grafton, MA 01536, USA.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Tremblay
authors.forename: Chueh-Ling
authors.initials: CL
authors.lastname: Kuo
authors.forename: Claudia
authors.initials: C
authors.lastname: Abeijon
authors.forename: Jorge
authors.initials: J
authors.lastname: Sepulveda
authors.forename: George
authors.initials: G
authors.lastname: Oyler
authors.forename: Xuebo
authors.initials: X
authors.lastname: Hu
authors.forename: Moonsoo M
authors.initials: MM
authors.lastname: Jin
authors.forename: Charles B
authors.initials: CB
authors.lastname: Shoemaker
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000494:pharmacology; D001666:Binding Sites, Antibody / Q000276:immunology; D001905:Botulinum Toxins / Q000032:analysis / Q000276:immunology*; D002161:Camelids, New World / Q000502:physiology*; D003014:Clostridium botulinum / Q000201:enzymology* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D020128:Inhibitory Concentration 50; D009474:Neurons; D009498:Neurotoxins / Q000032:analysis / Q000276:immunology*; D010447:Peptide Hydrolases / Q000276:immunology / Q000378:metabolism*; D019151:Peptide Library; D011480:Protease Inhibitors / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D009498:Neurotoxins; D019151:Peptide Library; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D001905:Botulinum Toxins