Blais Normand, Gagné Martin, Hamuro Yoshitomo, Rheault Patrick, Boyer Martine, Steff Ann-Muriel, Baudoux Guy, Dewar Vincent, Demers Josée, Ruelle Jean-Louis, Martin Denis
GSK Vaccines, Laval, Canada
GSK Vaccines, Laval, Canada.
J Virol. 2017 Jun 9;91(13). doi: 10.1128/JVI.02437-16. Print 2017 Jul 1.
The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen. Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.
人呼吸道合胞病毒（hRSV）融合（F）蛋白被认为是hRSV中和抗体反应的主要靶点。在病毒与宿主细胞膜融合时，这种糖蛋白会经历从预融合（pre-F）状态到融合后（post-F）状态的重大结构转变。最近的证据表明，与post-F状态相比，pre-F状态是中和抗体的更优靶点。因此，出于疫苗研发目的，我们设计并表征了一种重组hRSV F蛋白，称为Pre-F-GCN4t，其稳定在pre-F构象。为了证明Pre-F-GCN4t不会转变为post-F构象，将其与一种重组post-F分子Post-F-XC进行了比较。如化学变性所示，Pre-F-GCN4t被糖基化且为三聚体，其构象稳定性与Post-F-XC不同。电子显微镜分析表明Pre-F-GCN4t呈现出棒棒糖样结构。相比之下，Post-F-XC具有典型的细长锥形形状。氢/氘交换质谱表明这两种分子具有共同的刚性折叠核心和动态区域，并为它们的生物物理和生化特性及反应性提供了结构见解。结果表明，Pre-F-GCN4t比Post-F-XC更有效地从人血清中消耗hRSV中和抗体。重要的是，Pre-F-GCN4t还被证明能结合D25，一种对pre-F构象具有高度特异性的高效单克隆抗体。总之，这种构建体呈现出几个pre-F特征，不会转变为post-F构象，并呈现出保护性中和抗体反应所需的抗原特性。因此，Pre-F-GCN4t可被视为一种有前景的候选疫苗抗原。人呼吸道合胞病毒（RSV）是全球婴儿死亡和成人发病的主要原因。开发一种安全有效的RSV疫苗仍然是一个重要目标。RSV I类融合（F）糖蛋白被认为是最有前景的疫苗候选物之一，最近的证据表明预融合（pre-F）状态是中和抗体的更优靶点。我们的研究展示了Pre-F-GCN4t的物理化学特性，该分子设计为稳定在pre-F构象。为了确认其pre-F构象，将Pre-F-GCN4t与处于post-F构象的分子Post-F-XC进行了平行分析。我们的结果表明Pre-F-GCN4t呈现出稳定的pre-F构象特征，并支持将其用作RSV疫苗抗原。这样一种抗原可能代表了RSV疫苗研发的一项重大进展。
