Abstract
Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.
Highlights
Botulism is a flaccid paralysis caused by exposure to Botulinum neurotoxin (BoNT) that results primarily from ingestion of contaminated foods, the risk of exposure through deliberate events is considered sufficiently high to list BoNT as a Category A Priority Pathogen
We report the use of a recombinant, replicationincompetent, human Ad serotype 5 (Ad5) vector that promotes de novo secretion of antitoxin VHH-based neutralizing agents (VNAs) into serum
We demonstrate that a single treatment with Ad vector containing the VNA/ABP transgene (Ad/VNA)-BoNTA protects mice for several months from subsequent Botulinum neurotoxin serotype A1 (BoNT/A1) challenge, yet acts sufficiently rapid to be effective when administered shortly after toxin exposure
Summary
Botulism is a flaccid paralysis caused by exposure to Botulinum neurotoxin (BoNT) that results primarily from ingestion of contaminated foods, the risk of exposure through deliberate events is considered sufficiently high to list BoNT as a Category A Priority Pathogen. Toxin exposure is commonly treated by administration of antitoxin serum, generally prepared from large animals immunized with inactivated toxin [1,2,3]. Such antiserum products possess safety risks and are difficult to develop, produce and maintain. Antiserum is not practical for prophylactic protection of people that are considered at-risk of toxin exposures. We have reported the use of an alternative antitoxin strategy [5] which employs ‘VHH-based neutralizing agents’ (VNAs) consisting of linked 14 kDa camelid heavy-chain-only VH domains (VHHs), produced as heteromultimers, that bind and neutralize toxin targets. Coadministration of the anti-tag mAb, called the effector Ab (efAb), enhances therapeutic efficacy in some toxin models [5,6,7], probably by promoting toxin clearance through the liver [8]
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