Diesterbeck Ulrike S, Ahsendorf Henrike P, Frenzel André, Sharifi Ahmad Reza, Schirrmann Thomas, Czerny Claus-Peter
Division of Microbiology and Animal Hygiene, Department of Animal Sciences, University of Göttingen, Burckhardtweg 2, 37077 Göttingen, Germany.
Yumab GmbH, Science Campus Braunschweig Sued, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Vaccines (Basel). 2021 Nov 10;9(11):1308. doi: 10.3390/vaccines9111308.
A panel of potent neutralizing antibodies are protective against orthopoxvirus (OPXV) infections. For the development of OPXV-specific recombinant human single-chain antibodies (scFvs), the IgG repertoire of four vaccinated donors was amplified from peripheral B-lymphocytes. The resulting library consisted of ≥4 × 10 independent colonies. The immuno-screening against vaccinia virus (VACV) Elstree revealed a predominant selection of scFv clones specifically binding to the D8 protein. The scFv-1.2.2.H9 was engineered into larger human scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 formats to improve the binding affinity and to add effector functions within the human immune response. Similar binding kinetics were calculated for scFv-1.2.2.H9 and scFv-Fc-1.2.2.H9 (1.61 nM and 7.685 nM, respectively), whereas, for IgG1-1.2.2.H9, the Michaelis-Menten kinetics revealed an increased affinity of 43.8 pM. None of the purified recombinant 1.2.2.H9 formats were able to neutralize VACV Elstree in vitro. After addition of 1% human complement, the neutralization of ≥50% of VACV Elstree was achieved with 0.0776 µM scFv-Fc-1.2.2.H9 and 0.01324 µM IgG1-1.2.2.H9, respectively. In an in vivo passive immunization NMRI mouse model, 100 µg purified scFv-1.2.2.H9 and the IgG1-1.2.2.H9 partially protected against the challenge with 4 LD VACV Munich 1, as 3/6 mice survived. In contrast, in the scFv-Fc-1.2.2.H9 group, only one mouse survived the challenge.
一组强效中和抗体可预防正痘病毒（OPXV）感染。为了开发OPXV特异性重组人单链抗体（scFv），从四名接种疫苗供体的外周B淋巴细胞中扩增IgG文库。所得文库包含≥4×10个独立菌落。针对痘苗病毒（VACV）Elstree的免疫筛选显示，主要筛选出了与D8蛋白特异性结合的scFv克隆。将scFv-1.2.2.H9构建成更大的人scFv-Fc-1.2.2.H9和IgG1-1.2.2.H9形式，以提高结合亲和力并在人体免疫反应中添加效应功能。计算得出scFv-1.2.2.H9和scFv-Fc-1.2.2.H9的结合动力学相似（分别为1.61 nM和7.685 nM），而对于IgG1-1.2.2.H9，米氏动力学显示亲和力增加至43.8 pM。纯化的重组1.2.2.H9形式均不能在体外中和VACV Elstree。加入1%人补体后，分别用0.0776 µM scFv-Fc-1.2.2.H9和0.01324 µM IgG1-1.2.2.H9实现了对≥50% VACV Elstree的中和。在体内被动免疫NMRI小鼠模型中，100 µg纯化的scFv-1.2.2.H9和IgG1-1.2.2.H9对4个致死剂量的VACV慕尼黑1株攻击有部分保护作用，因为有3/6的小鼠存活。相比之下，在scFv-Fc-1.2.2.H9组中，只有1只小鼠在攻击后存活。
