Abstract
In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that the BoNT-neutralizing potency of mAbs can be improved through red blood cell (RBC) immunoadherence. For example, a fusion protein (FP) that adheres biotinylated mAbs to the RBC surface enabled a pair of mAbs to neutralize 5000 LD50 BoNT/A in the mouse protection assay. Here, we added two mAbs to that combination, creating a 4-mAb:FP complex that neutralized 40,000 LD50 BoNT/A in vivo, and analyzed functional correlates of neutralization. The FP enhanced potency of BoNT/A immune complexes, providing the greatest magnitude of benefit to the 4-mAb combination. RBC binding of a BoNT/A complexed with 4-mAb:FP exhibited a bi-phasic clearance process in vivo. Most of the complexes were cleared within five minutes; the rest were cleared gradually over many hours. Peritoneal macrophages showed better uptake of the 4-mAb complex than the 3-mAb complex, and this was not affected by the presence of the FP. However, the addition of RBCs to the 4-mAb:FP BoNT/A doubled macrophage uptake of the complexes. Lastly, the 4-mAb:FP BoNT/A complex synergistically induced M2 macrophage polarization, as indicated by IL-10 expression, whether or not RBCs were present. RBC-targeted immunoadherence through the FP is a potent enhancer of mAb-mediated BoNT/A neutralization in vivo, and can have positive effects on BoNT/A sequestration, immune complex uptake, and macrophage activation.
Highlights
Despite widespread progress in antimicrobial drug development, emerging infectious diseases, drug-resistant pathogens, and toxin-producing pathogens remain a considerable global concern.Monoclonal antibodies have proven to be safe and effective as therapeutics for treating infectious diseases and toxins, yet their broad potential has been largely unrealized
We sought additional Monoclonal antibodies (mAbs) that would enable us to study the impact of higher-order complexes on red blood cell (RBC)-dependent botulinum neurotoxin (BoNT)/A neutralization and clearance in vivo
Oligoclonal mAbs can provide some of the benefits of the authentic polyclonal antiantibody response, including enhanced inhibition of toxin activity, improved binding affinity, alteration toxin antibody response, including enhanced inhibition of toxin activity, improved binding affinity, of toxin conformation, and improved clearance in vivo [22,23,24,25]
Summary
Monoclonal antibodies (mAbs) have proven to be safe and effective as therapeutics for treating infectious diseases and toxins, yet their broad potential has been largely unrealized. This is partly because single mAbs have limited utility against pathogens that are antigenically diverse, have redundant mechanisms of virulence, or require oligomeric immune complex formation for clearance. Combinations of mAbs that have complementary binding and neutralizing activities may be able to circumvent these problems. We have been studying immune complex mediated clearance in vivo using neutralization of botulinum neurotoxin (BoNT) as a model system. BoNT is the most potent biological toxin known, and is ranked by CDC as a Category A Select
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