Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein
Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein
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DOI：
10.1371/journal.pone.0017491
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发表时间：
2011-03-02
期刊：
影响因子：
3.7
通讯作者：
Dessain, Scott K.
中科院分区：
文献类型：
--
作者：
Adekar, Sharad P.;Segan, Andrew T.;Dessain, Scott K.
肉毒杆菌神经毒素(BoNT)能有效地抑制神经肌肉接头的胆碱能信号。接触BONT的理想对策是单抗或BONT抗血清，它们形成含有BONT的免疫复合体，可迅速从一般循环中清除。调理毒素的清除可能涉及补体受体介导的免疫黏附灵长类动物的红细胞(RBC)或啮齿动物的血小板。增强BONT特异性抗体的免疫粘附性的方法可能会增加它们在体内的效力。我们设计了一种新型的融合蛋白(FP)，将生物素化的分子与红细胞表面的血糖蛋白A(GPA)连接起来。FP由小鼠GPA特异的单链抗体与链霉亲和素融合而成。FP：MAB：BONT复合体在体外与红细胞表面特异性结合。在BONT中和的小鼠模型中，FP增加了单抗和双抗体组合在BONT中和中和的效力。两种抗体与FP结合后，在小鼠体内完全中和了5,000个LD50 BoNT。体内中和依赖于两种抗体的生物素化，并与血浆BONT水平的降低相关。在暴露后中毒模型中，FP：MAB复合体在毒素暴露后2小时内给予完全保护，使其免受致命性BONT/A1剂量的伤害。在暴露前预防模型中，小鼠在注射FP：MAb复合体后72小时内得到完全保护。这些结果表明，通过FP的RBC靶向免疫黏附是体内抗体中和BONT的有效增强剂。
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.