A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene
A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene
复制标题
DOI：
10.1128/jvi.03473-14
复制
发表时间：
2015-03-01
影响因子：
5.4
通讯作者：
Moore, John P.
中科院分区：
文献类型：
--
作者：
Pugach, Pavel;Ozorowski, Gabriel;Moore, John P.
人免疫缺陷病毒1型（HIV-1）包膜糖蛋白（Env）刺突的重组三聚体模拟物应暴露尽可能多的广泛中和抗体（bNAb）表位，但很少（如果有的话）暴露非中和抗体（non-NAb）表位。基于亚型A菌株BG 505的可溶性切割的SOSIP.664 gp 140三聚体接近这一理想，因此是合理的疫苗候选物。在这里，我们报告的生产和体外特性的一个新的SOSIP。664三聚体来自亚型B env基因，B41，包括如何使这种蛋白质在低血清介质中没有蛋白水解损伤（剪切）的V3区。我们还表明，nonclipped三聚体可以成功地纯化通过阳性选择亲和柱使用的bNAb PGT 145，它承认一个四级结构依赖的表位在三聚体顶点。负染电子显微镜成像显示，纯化的，非剪切的，天然样B41 SOSIP。664三聚体包含两个亚群，我们建议代表完全封闭和更开放的构象之间的平衡。后者不同于完全开放的CD 4受体结合构象，可能代表三聚体的中间状态。这种新的亚型B三聚体添加到适合免疫原性和结构研究的天然样Env蛋白的库中。重要信息切割的三聚体包膜蛋白复合物是HIV-1表面上唯一的中和抗体靶标。许多疫苗策略基于诱导中和抗体。对于HIV-1，一种方法涉及使用天然三聚体的重组可溶性蛋白质模拟物。目前，以实际量制备天然样可溶性三聚体的唯一可靠方法是通过引入赋予Env切割形式稳定性的特异性序列变化。得到的蛋白质被称为SOSIP.664 gp 140三聚体，目前的范例是基于BG 505亚型A env基因。在这里，我们描述的生产和表征的SOSIP. 664蛋白来自亚型B基因（B41），连同一个简单的，一步的方法来纯化天然样三聚体通过亲和层析与三聚体特异性bNA B，PGT 145。由此产生的三聚体将用于旨在设计制造有效HIV-1疫苗的方法的结构和免疫原性实验。
Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP. 664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.IMPORTANCEThe cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.