Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Retrovirology. 2010 Feb 16;7:11. doi: 10.1186/1742-4690-7-11.
We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.
We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.
The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.
我们之前描述了一种有效的重组 HIV-1 中和蛋白 sCD4-17b，它由通过柔性多肽接头连接的可溶性 CD4 组成，连接到针对 gp120 中高度保守的 CD4 诱导桥接片的 17b 人源单克隆抗体的 scFv。双功能蛋白的 sCD4 部分与游离病毒上的 gp120 结合，从而使 17b scFv 部分能够结合并阻断 gp120/辅助受体相互作用，这是进入所必需的。使用 MAGI-CCR5 测定系统的先前研究表明，sCD4-17b（在浓缩细胞培养液中或部分纯化）能够有效地中和几种遗传上不同的 HIV-1 原发性分离物；然而，尽管 CD4 和 17b 的结合位点都保守，在测试的浓度下，它对其他几种菌株都没有效果。为了解决这个难题，我们设计了具有不同接头长度的 sCD4-17b 变体，并使用 TZM-blEnv 假型测定系统，在更广泛的浓度范围内，针对大量遗传上不同的 HIV-1 原发性分离物，测试免疫亲和纯化蛋白的中和活性。我们还检查了源自不同产生细胞类型的同基因病毒对 sCD4-17b 的敏感性。
我们观察到，免疫亲和纯化的 sCD4-17b 能够有效地中和 HIV-1 假型，包括在 MAGI-CCR5 测定中先前发现相对不敏感的 HIV-1 分离物。在假型颗粒和传染性病毒粒子中，观察到原始构建体和具有较长接头的变体的效力相当；相比之下，接头太短以至于不能同时结合 sCD4 和 17b scFv 部分的构建体的效力要低得多。sCD4-17b 对来自不同遗传亚型（A、B、C、D、F 和循环重组形式 AE 和 AG）的近 40 种 HIV-1 原发性分离物的 100%具有强大的中和活性。其中和广度和效力优于广泛中和的单克隆抗体 IgG b12、2G12、2F5 和 4E10。sCD4-17b 的活性被发现与源自转染产生细胞系的传染性分子克隆或仅通过 PBMC 传代一次的同基因病毒颗粒相似；这与单克隆抗体形成对比，后者对 PMBC 传代的病毒的效力较低。
结果突出了 sCD4-17b 对遗传上不同的 HIV-1 原发性分离物的极其强大和广泛的中和活性。该双功能蛋白在对抗 HIV 感染的抗病毒方法中有潜在的应用。
