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首页> 外文期刊>Journal of Molecular Biology >Characterization and HIV-1 Fusion Inhibitory Properties of Monoclonal Fabs Obtained From a Human Non-immune Phage Library Selected Against Diverse Epitopes of the Ectodomain of HIV-1 gp41.
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Characterization and HIV-1 Fusion Inhibitory Properties of Monoclonal Fabs Obtained From a Human Non-immune Phage Library Selected Against Diverse Epitopes of the Ectodomain of HIV-1 gp41.

机译：从人类非免疫噬菌体库中获得针对HIV-1 gp41的电子域的不同表位的单克隆抗体的表征和HIV-1融合抑制特性。

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Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35(CCG)-N13, N34(CCG) (a smaller version of N35(CCG)-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC(50) values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6mug/ml to 60mug/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.
机译：使用以Fab格式包含超过10（9）个功能性人抗体特异性的人非免疫噬菌体文库，我们已经能够从HIV-1中选择针对gp41胞外域的多种表位的八种单克隆Fab。用于淘选抗体的抗原包含两个衍生自gp41，N（CCG）-gp41和N35（CCG）-N13的可溶性二硫键连接的三聚体多肽。前者包含gp41的N螺旋的裸露三聚螺旋螺旋，螺旋螺旋状融合到gp41的最小热稳定胞外域，而后者仅包含N螺旋的三聚螺旋螺旋。通过Western印迹分析针对四种抗原的探针被选出：N（CCG）-gp41，N35（CCG）-N13，N34（CCG）（N35（CCG）-N13的较小版本）和最小的热稳定胞外域核心gp41的六螺旋束构象（6-HB）。发现三类Fab：A类（两个Fab）主要与6-HB相互作用。 B类（四个Fab）与6 HB和N螺旋的内部三聚体螺旋线圈相互作用; C类（两个Fab）与N螺旋的内部三聚体螺旋线圈特异性相互作用。 Fabs的IC（50）值表示为二价微型抗体，在基于牛痘病毒的报告基因检测HIV-1包膜介导的细胞融合中，使用来自HIV-1 T热带毒株LAV。两种最有效的融合抑制剂属于B类。这组Fab提供了一组有用的探针，可用于研究HIV-1包膜介导的细胞融合，并可作为开发基于Fab的抗HIV-1治疗剂的基础。

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