HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测
Summary
Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.
Abstract
HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.
Introduction
人类免疫缺陷病毒1型（HIV-1）的条目，以三聚体的病毒包膜糖蛋白（环境）介导的感染周期的第一步骤。是唯一外露的病毒抗原，在病毒粒子的表面呈现，将信封三聚体引发中和非中的抗体。因此，它表示用于疫苗的免疫原设计了一个有趣的候选者。然而，信封疫苗试验中的可溶性或重组的形式引起的反应，只有对最初级的HIV-1分离株1-3最小的有效性。尽管如此，部分疗效的HIV-1包膜的RV144疫苗试验4新的兴趣作为免疫原的候选观察。这是证实了最近的一项研究说明了疫苗引起的抗包膜抗体均足以产生一定程度的保护，防止SIV和HIV挑战5。
后所要合成的内质网中，包膜糖蛋白在前驱物的gp160，经历，这对于其对燃料的病毒融合过程能力的关键不同的翻译后修饰。被裂解成其特细胞质gp120和gp41的跨膜亚基6-10，与非共价相互作用维持的gp120-gp41的联系之前，信封前体必须正确和准倍三聚体。受感染的细胞机器还负责大量糖基化的包膜，其包含约50％的总质量为11,12。所得到的复合结构允许信封是构象柔性13,14，同时提供了被认为是允许信封，以适应和隐藏某些高度免疫原性表位，否则将暴露15-19亚稳态，突出的重要性，以更好地理解不同的构象由天然包膜三聚体取样。
迄今为止，一些技术已经开发并成功地用于研究包膜构象沟道安格斯。然而，他们在他们的局限性会发生变化，而通常被限制在特定的包膜上下文。例如，表面等离子体共振，或使用构象特异性单克隆抗体的免疫沉淀实验（单抗），依赖于任一的单体的水溶性或可溶的信封分子是已知的，从它们的三聚体形式的20,21 immunogenetically不同。最近的研究还表明，裂解影响导致主要抗原决定簇所认可非中抗体14,22,23曝光信封构象。
在这里，我们详细描述了一种方法，它允许快速和容易的决心cellularly表达信封三聚体18,24-26的构象。下列信封的人贴壁细胞系的瞬时转染包膜特异性抗体的结合使用了一个简单的化学发光反应进行检测。这种技术也可以用来表征的构象偏好构象依赖新生凹痕抗体。因此，这个实验提供了一个强大且高度灵活的检测方法。
Protocol
Representative Results
Discussion
该测定被优化以检测特异性单克隆抗体的相互作用与HIV-1三聚体包膜表达在细胞表面。一旦该协议已经建立，它可用于在中到高吞吐量和低的总材料成本和抗体的小的量。由于该测定是染为基础，它可以很容易地以研究它们对包膜构象效应适于胞内蛋白如CD4的共表达。
然而，该协议的转基也意味着，这是其最重要的缺陷之一。首先，抗原被研究这种技术是必需的，以提供在一…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢詹姆斯·罗宾逊博士的慷慨的A32，17B，48D的礼物，和C11单克隆抗体。 PGT 121通过美国国立卫生研究院艾滋病试剂项目，艾滋病，NIAID，美国国立卫生研究院（目录号12343）中科获得。这项工作是由加拿大创新基金会项目负责人＃29866的支持，由CIHR的操作＃257792，由FRQS设立的青年科学家给予＃24639到自动对焦，并通过CRCHUM连续补助金以及由CIHR资助的催化剂＃126630房颤和MR。房颤是一种FRSQ Chercheur布西尔少年团契1＃24639的收件人。 MV由CIHR博士后研究奖＃291485支持。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| HOS cells | ATCC | CRL-1543 | |
| White Opaque Tissue Culture Plate, 96 well, Flat Bottom | BD | 353296 | |
| Polyethylenimine, linear, 25000 MW | Polysciences | 23966 | Prepared in 1mg/ml solution |
| Dulbecco's Modified Eagle Medium | Invitrogen | 11995 | |
| Goat Anti-Human IgG, Peroxidase Conjugated | Pierce | 31413 | |
| Enhanced Chemiluminescence Substrate | PerkinElmer | NEL105001EA | |
| TriStar LB 941, Plate Reader | Berthold Technologies |
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