Song Qiaoyun, Yang Haixia, Zhu Haoyue, Hu Yun, Shen Wenling, Cheng Huifeng, Cai Jialiao, Qiu Manlan, Li Yueyue, Li Yaolan, Ye Wencai, Wang Ying, Tang Wei
State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University, Guangzhou, People's Republic of China.
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM & New Drugs Research, Guangdong-Hong Kong-Macau Joint Laboratory for Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou, People's Republic of China.
J Virol. 2025 Jun 17;99(6):e0008725. doi: 10.1128/jvi.00087-25. Epub 2025 May 28.
Respiratory syncytial virus (RSV) entry into host cells is facilitated by viral fusion, wherein the metastable RSV fusion (F) protein undergoes a conformational change from a prefusion state to a highly stable postfusion structure. The prefusion F elicits a more robust human antibody response than its postfusion F and is a primary target for RSV vaccine development. However, the inherent instability of the prefusion F trimer and its low protein expression level in host cells are a significant challenge for developing a high-potency RSV vaccine. Here, we report that the introduction of four hydrophobic residue substitutions in the RSV F protein resulted in a highly stable prefusion F trimer (pre-F-IFLP). This engineered variant exhibits enhanced expression and stability compared to DS-Cav1, with improved thermal stability, increased resistance to acid and base, and extended storage life. Furthermore, pre-F-IFLP induced neutralizing antibody responses 72-fold higher than those elicited by DS-Cav1 following a second booster immunization and fully protected mice against RSV infection.
In this study, we demonstrate that introducing four hydrophobic residue substitutions into the RSV F protein leads to the generation of a highly stable prefusion F trimer (pre-F-IFLP) with improved expression levels in cultured cells and superior stability compared to DS-Cav1, the first-generation prefusion F-stabilized RSV vaccine. Furthermore, pre-F-IFLP induced significantly higher neutralizing antibody responses than DS-Cav1 following both the first and second booster immunizations and conferred complete protection against RSV infection in a mouse model. These findings present an alternative approach for stabilizing the trimeric prefusion F protein, enhancing its expression, and significantly improving its protective efficacy for the prevention of RSV infection .
呼吸道合胞病毒（RSV）通过病毒融合进入宿主细胞，其中亚稳态的RSV融合（F）蛋白经历从预融合状态到高度稳定的融合后结构的构象变化。与融合后的F相比，预融合F引发更强的人类抗体反应，是RSV疫苗开发的主要靶点。然而，预融合F三聚体固有的不稳定性及其在宿主细胞中的低蛋白表达水平是开发高效RSV疫苗的重大挑战。在此，我们报告在RSV F蛋白中引入四个疏水残基取代导致形成高度稳定的预融合F三聚体（pre-F-IFLP）。与DS-Cav1相比，这种工程变体表现出增强的表达和稳定性，具有改善的热稳定性、增加的耐酸碱能力和延长的储存寿命。此外，在第二次加强免疫后，pre-F-IFLP诱导的中和抗体反应比DS-Cav1引发的反应高72倍，并能完全保护小鼠免受RSV感染。
在本研究中，我们证明在RSV F蛋白中引入四个疏水残基取代可导致产生高度稳定的预融合F三聚体（pre-F-IFLP），其在培养细胞中的表达水平提高，与第一代预融合F稳定的RSV疫苗DS-Cav1相比具有更高的稳定性。此外，在第一次和第二次加强免疫后，pre-F-IFLP诱导的中和抗体反应均显著高于DS-Cav1，并在小鼠模型中提供了针对RSV感染的完全保护。这些发现为稳定三聚体预融合F蛋白、增强其表达并显著提高其预防RSV感染的保护效力提供了一种替代方法。
