A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes
A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes
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DOI：
10.1371/journal.pone.0174187
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发表时间：
2017-03-21
期刊：
影响因子：
3.7
通讯作者：
Marks, James D.
中科院分区：
文献类型：
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作者：
Fan, Yongfeng;Garcia-Rodriguez, Consuelo;Marks, James D.
人类肉毒杆菌中毒主要由 A、B 和 E 血清型肉毒杆菌神经毒素 (BoNT) 引起，约 1% 由 F 血清型 (BoNT/F) 引起。 BoNT/F包含至少7种不同的亚型，亚型之间的氨基酸序列差异高达36%。序列差异对生成能够结合、检测和中和所有 BoNT/F 亚型的单克隆抗体 (mAb) 提出了重大挑战。我们使用免疫小鼠抗体可变 (V) 区的库克隆和酵母展示来生成一组 33 个先导单链 Fv (scFv) mAb，它们结合一种或多种 BoNT/F 亚型，中位平衡解离常数 (K-D) 为 4.06 x 10(-9) M。通过使先导 mAb 的 V 区多样化并选择交叉反应性，我们生成了 5 个 mAb，可结合七种中的每一种。亚型。三个scFv结合非重叠表位被转化为IgG，其对于不同BoNT/F亚型的KD范围为2.2x10(-8) M至1.47x10(-12) pM。在小鼠中和试验中，等摩尔的 mAb 组合能够有效中和 BoNT/F1、F2、F4 和 F7。单克隆抗体具有潜在的实用价值，能够识别已知的 BoNT/F 亚型，并可开发为抗毒素来预防和治疗 F 型肉毒杆菌中毒。
Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (K-D) of 4.06 x 10(-9) M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2x10(-8) M to 1.47x10(-12) pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.