Background: Influenza remains a significant global health burden, causing 3-5 million severe cases and 290000-650000 deaths annually. Current influenza vaccines face several limitations, including suboptimal efficacy (ranging from 10 to 60%), strain mismatches due to antigenic drift, and annual administration, highlighting the need for improved vaccination strategies. In this context, self-amplifying RNA (saRNA) are a promising approach for vaccine development, offering enhanced immunogenicity through sustained antigen expression while requiring lower doses and maintaining favorable safety profiles. Rhinoviruses, with their well-characterized replication machinery and tropism for respiratory epithelium, represent a novel strategy for developing saRNA-based vaccines targeting respiratory pathogens such as influenza virus.
Methods: We developed rhinovirus replicons by deleting structural protein-encoding sequences from the viral genome and preserving essential replication elements including the 5' and 3' untranslated regions and non-structural protein genes (i.e. saRNA). The deleted structural protein regions were replaced with either reporter genes—eGFP and nLuc—for functional validation, or with influenza hemagglutinin gene for immunological studies and vaccine applications. Rhinovirus replicon activity was assessed through saRNA transfection in cell lines and subsequent measurement of replication capacity, protein expression levels, and cellular viability through flow cytometry, fluorescence microscopy, and luminescence assays.
Results: Rhinovirus saRNAs demonstrated strong self-replication capacity when structural proteins were replaced with reporter genes, confirming the preservation of viral replication machinery. Reporter gene expression was successfully detected and sustained over extended periods (up to 72 hours) in several cell lines, indicating efficient RNA amplification and versatility of the system. Studies also revealed higher replication efficiency of saRNA compared to mRNA, demonstrating enhanced and prolonged protein expression, suggesting potentially enhanced antigen expression and immunogenicity compared to traditional mRNA approaches. saRNA replication reduced cell viability, possibly improving immunity through antigen presenting cell recruitment and activation. These findings demonstrate the capacity of this platform in supporting different genes while preserving essential functions for RNA replication and protein expression.
Conclusion: This study establishes rhinovirus-based saRNA as a novel and viable approach for influenza vaccine development. The successful replacement of structural proteins with genes of interest while maintaining replication competency in different tissues demonstrates the efficiency and flexibility of this system. The respiratory tropism of rhinoviruses may provide additional advantages for inducing mucosal immunity against respiratory pathogens such as influenza. Future work will focus on evaluating immunogenicity in animal models and exploring delivery mechanisms for clinical translation.