Zhang Mei-Yun, Choudhry Vidita, Sidorov Igor A, Tenev Vladimir, Vu Bang K, Choudhary Anil, Lu Hong, Stiegler Gabriela M, Katinger Hermann W D, Jiang Shibo, Broder Christopher C, Dimitrov Dimiter S
Protein Interactions Group, CCRNP, CCR, NCI-Frederick, NIH, Frederick, Maryland 21702, USA.
J Immunol Methods. 2006 Dec 20;317(1-2):21-30. doi: 10.1016/j.jim.2006.09.016. Epub 2006 Oct 16.
The HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against Env-based antigens has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a novel gp41-specific human monoclonal antibody (hmAb), m48, from an immune library derived from long-term nonprogressors with high titers of broadly cross-reactive neutralizing antibodies (bcnAbs). Selection of m48 was only successful using CAP and not through the conventional pre-incubation methodology. In assays based on spreading infection in peripheral blood mononuclear cells (PBMCs) m48 neutralized a panel of HIV-1 primary isolates from different clades more potently than the well-characterized broadly cross-reactive HIV-1-neutralizing antibodies IgG1 4E10 and Fab Z13. These results may have implications for the selection of novel gp41-specific bcnAbs and other antibodies, and for the development of HIV-1 inhibitors and vaccine immunogens.
人类免疫缺陷病毒包膜糖蛋白（Env）由两个非共价结合的亚基组成：gp120和gp41。针对基于Env的抗原进行噬菌体展示抗体文库淘选，大多筛选出了抗gp120抗体。在没有gp120的情况下，天然gp41不稳定。使用gp41片段作为抗原，只能筛选出中和活性相对较低的抗体。为了在完整Env背景下增强对gp41特异性抗体的筛选，我们开发了一种称为竞争性抗原淘选（CAP）的方法。利用CAP，我们从具有高滴度广泛交叉反应性中和抗体（bcnAbs）的长期非进展者来源的免疫文库中，鉴定出一种新型的gp41特异性人单克隆抗体（hmAb）m48。只有使用CAP才能成功筛选出m48，而通过传统的预孵育方法则无法成功筛选。在外周血单核细胞（PBMC）中基于传播感染的检测中，m48比特征明确的广泛交叉反应性HIV-1中和抗体IgG1 4E10和Fab Z13更有效地中和了一组来自不同分支的HIV-1原代分离株。这些结果可能对新型gp41特异性bcnAbs和其他抗体的筛选，以及对HIV-1抑制剂和疫苗免疫原的开发具有启示意义。
