Cavity-Filling Mutations as a Strategy to Stabilize Prefusion Viral Fusion Proteins for Vaccine Design

Enveloped viruses use metastable fusion proteins to enter host cells through large-scale conformational rearrangements. The prefusion conformation represents a higher-energy metastable state that irreversibly refolds into a lower-energy postfusion conformation during membrane fusion. Although the prefusion state contains the key neutralizing epitopes, it represents an ideal target for vaccine development. However, because of the instability of the prefusion state, the development of a viable vaccine and the determination of the prefusion state are both major challenges. Building on advances in structure-based vaccine design, cavity-filling mutations have emerged as a promising strategy for stabilizing prefusion viral fusion proteins across viral systems. Cavity-filling mutations have been shown in several viral systems to improve protein stability, thermostability, antigen expression, and immunogenicity. However, these effects depend on the structural architecture, cavity geometry, and local conformational context of each protein. In addition, these modifications may reduce premature transitions to the postfusion conformation and may help preserve critical neutralizing epitopes, depending on the structural context, while maintaining a metastable prefusion state. Accordingly, this review summarizes the molecular basis of cavity-filling-mediated stabilization, highlights successful applications across diverse viral families, examines how structural stabilization influences immunogenicity, and discusses the potential of cavity-filling strategies for rational vaccine design.