Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelope glycoprotein.
Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelope glycoprotein.
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建立一种检测HIV-1包膜糖蛋白的RPLC-UV方法。
DOI：
10.1039/d1ay00072a
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发表时间：
2021-05-21
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HIV-1 疫苗设计工作之一的重点是开发重组 HIV-1 三聚体包膜糖蛋白 (Env) 作为免疫原来诱导广泛中和抗体。 BG505.DS.SOSIP.664 gp140 (Env) 构建体是一种类似天然的免疫原，已被充分表征为候选疫苗。该候选疫苗由三个相同的 gp120 和截短的 gp41 亚基组成，形成异二聚体三聚体。在生产过程中，重组 Env 表达为 gp140 前体多肽，其中弗林蛋白酶可切割位点被改造以产生 gp120 和 gp41 亚基的异二聚体。每个异二聚体通过分子间二硫键连接，三个异二聚体形成三聚体。弗林蛋白酶裂解是模仿天然 HIV-1 Env 构象的重要因素，并且有助于诱导免疫反应。因此，监测裂解对于确保 Env 疫苗产品的功能至关重要。本文开发了一种结合还原的新 RPLC-UV 方法，用于常规测定未切割的 gp140 相对于切割的 gp120 和 gp41 亚基的百分比。在 gp120、gp41 和未切割的 gp140 峰之间实现了基线分离，从而实现了未切割的 gp140 的相对定量。总体而言，这种 RPLC-UV 方法已成功应用于支持 Env 候选疫苗的开发。我们展示了一种定量且灵敏的 RPLC-UV 策略，并结合减少在 HIV-1 疫苗开发和制造过程中常规监测重组包膜糖蛋白构建体的弗林蛋白酶裂解效率。
One of the HIV-1 vaccine design efforts has focused on developing a recombinant HIV-1 trimeric envelope glycoprotein (Env) as an immunogen to induce broadly neutralizing antibodies. A native-like immunogen, the BG505.DS.SOSIP.664 gp140 (Env) construct has been well-characterized as a vaccine candidate. This vaccine candidate comprises of three identical gp120 and truncated gp41 subunits that form into a trimer of heterodimers. During production, recombinant Env is expressed as a gp140 precursor polypeptide in which a furin cleavable site is engineered to generate a heterodimer of gp120 and gp41 subunits. Each heterodimer is connected by an intermolecular disulfide bond, and three heterodimers form into a trimer. Furin cleavage is an important factor to mimic native-like HIV-1 Env conformations and is needed to help induce an immune response. Therefore, it is critical to monitor cleavage for ensuring functionality of the Env vaccine product. In this paper, a new RPLC-UV method coupled with reduction was developed to routinely determine the percentage of uncleaved gp140 relative to the cleaved gp120 and gp41 subunits. Baseline separation was achieved among the gp120, gp41 and uncleaved gp140 peaks, thus enabling relative quantification of uncleaved gp140. Overall, this RPLC-UV approach has been successfully applied to support Env vaccine candidate developments. We demonstrate a quantitative and sensitive RPLC-UV strategy coupled with reduction to routinely monitor furin cleavage efficiency of recombinant envelope glycoprotein constructs during HIV-1 vaccine development and manufacturing.
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影响因子：
8.7
作者：
Sanders RW;Moore JP
通讯作者：
Moore JP
影响因子：
6.7
作者：
Sanders RW;Derking R;Cupo A;Julien JP;Yasmeen A;de Val N;Kim HJ;Blattner C;de la Peña AT;Korzun J;Golabek M;de Los Reyes K;Ketas TJ;van Gils MJ;King CR;Wilson IA;Ward AB;Klasse PJ;Moore JP
通讯作者：
Moore JP
影响因子：
4.8
作者：
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通讯作者：
Rao, Venigalla B.
影响因子：
5.4
作者：
Chuang, Gwo-Yu;Lal, Yen-Ting;Kwong, Peter D.
通讯作者：
Kwong, Peter D.
DOI：
10.1039/c9an02098e
发表时间：
2020-03-02
期刊：
The Analyst
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