title: Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.
abstract: Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.
authors.affiliation: Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, United States of America.
authors.forename: Sharad P
authors.initials: SP
authors.lastname: Adekar
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Segan
authors.forename: Cindy
authors.initials: C
authors.lastname: Chen
authors.forename: Rodney
authors.initials: R
authors.lastname: Bermudez
authors.forename: M D
authors.initials: MD
authors.lastname: Elias
authors.forename: Bernard H
authors.initials: BH
authors.lastname: Selling
authors.forename: B P
authors.initials: BP
authors.lastname: Kapadnis
authors.forename: Lance L
authors.initials: LL
authors.lastname: Simpson
authors.forename: Paul M
authors.initials: PM
authors.lastname: Simon
authors.forename: Scott K
authors.initials: SK
authors.lastname: Dessain
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000276:immunology*; D019921:Biotinylation; D019274:Botulinum Toxins, Type A / Q000097:blood / Q000276:immunology*; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D007267:Injections; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D011993:Recombinant Fusion Proteins; D019274:Botulinum Toxins, Type A