Hydrophobic residue substitutions enhance the stability and in vivo immunogenicity of respiratory syncytial virus fusion protein.

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. IMPORTANCE: 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 in vivo.