Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases.
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.
Tremblay
Jacqueline M
JM
Tufts Cummings School of Veterinary Medicine, Department of Biomedical Sciences, 200 Westboro Road, North Grafton, MA 01536, USA.
Kuo
Chueh-Ling
CL
Abeijon
Claudia
C
Sepulveda
Jorge
J
Oyler
George
G
Hu
Xuebo
X
Jin
Moonsoo M
MM
Shoemaker
Charles B
CB
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
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D009498:Neurotoxins; D019151:Peptide Library; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D001905:Botulinum Toxins