Jingjing Zou

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory disease, and stabilization of the prefusion (preF) conformation of the F glycoprotein is central for vaccine development. Here, we report a structure-guided engineering strategy that enhances preF stability by reducing the hydrophobic cavity within the trimeric F protein. Targeted modifications at metastability-associated sites generated RVF-88, a disulfide-free stabilized preF immunogen that preserves key neutralizing epitopes, including antigenic site Ø, while exhibiting improved structural integrity and long-term storage stability. Formulated as an unadjuvanted bivalent vaccine, RVF-88 elicited potent neutralizing antibody responses and durable immune protection lasting up to 5 months in mice. Vaccination also protected both mice and cotton rats against RSV challenge. Structural analyses confirmed the intended cavity-reduction design, revealing a reduced apical hydrophobic cavity volume and surface area while maintaining the prefusion architecture. Together, these findings establish hydrophobic cavity reduction as a rational strategy for stabilizing prefusion RSV F and provide a promising next-generation vaccine candidate with improved stability and immunogenicity for further clinical development.

Lonafarnib, an oral antiviral that targets the fusion glycoprotein of respiratory syncytial virus (RSV), has demonstrated efficacy in vitro and in vivo . However, because the RSV has evolved to become resistant to other fusion inhibitors, there is a concern that the same could occur for lonafarnib. Here, we identified resistance to lonafarnib in the RSV A2 strain and a recent clinical isolate, RSV ON1, via in vitro selection at scale. Cell‒cell fusion and recombinant live RSV analysis confirmed that the mutations at K394, K399, and T400 of the cysteine-rich region of the fusion protein mediate high-level resistance. Lonafarnib resistance mutations also confer cross-resistance to other fusion inhibitors of clinical interest. All-atom molecular dynamics simulations revealed that these resistance mutations confer reduced stability to the fusion protein, thereby diminishing its binding affinity with lonafarnib. To address this vulnerability proactively and increase the barrier to resistance development, we designed the first potent proteolysis-targeting chimera (PROTAC) fusion protein degrader, compound 0179841, which uses lonafarnib and cereblon as ligands. This PROTAC effectively inhibited RSV replication. Collectively, our findings indicate that RSV develops resistance to lonafarnib in the cysteine-rich region of the fusion protein. This work sheds light on the mechanisms by which RSV evolves resistance to lonafarnib and provides a foundation for the rational design of antivirals aimed at preventing resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection poses a substantial public health challenge. Resistance to several potent fusion inhibitors, which are currently in various stages of clinical development, can readily emerge. Through a drug repurposing screen, we identified lonafarnib as an RSV fusion inhibitor; however, concerns exist regarding the potential development of resistance. Here, large-scale in vitro selection experiments revealed specific mutations within the highly conserved cysteine-rich region of the fusion (F) protein that confer high-level lonafarnib resistance across diverse RSV strains. These resistance mutations also confer cross-resistance to other clinical-stage fusion inhibitors. Mechanistic investigations demonstrated that these mutations reduce F protein stability, thereby diminishing the binding affinity of lonafarnib. As a proof of concept for an alternative antiviral strategy, we rationally designed the first potent proteolysis-targeting chimera (PROTAC) F protein degrader, compound 0179841, by utilizing lonafarnib and cereblon ligands. This novel antiviral agent effectively inhibits RSV infection by inducing degradation of the F protein. This work elucidates the molecular basis of RSV resistance to lonafarnib and establishes a strategy for developing next-generation antivirals aimed at preempting resistance.