Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

. Furthermore, the transgene was easily swapped with a diverse array of genes encoding human IL-15, nanoluciferase, or fluorescent proteins such as miniSOG and miRFP670nano, highlighting the versatility of the platform. IMPORTANCE: IAV-ΔNS1 vectors are promising vaccine platforms that elicit strong immune responses with a good safety profile. However, integration of immune-stimulatory cytokines into vector design to boost immunogenicity has been technically challenging. In this study, we developed a genetically re-engineered segment 8 design that overcomes prior limitations due to design complexity or vector efficiency, enabling high-level expression of the transgene, i.e., human interleukin 2 and other diverse proteins. The platform retains strong replicative capacity in permissive systems and remains genetically stable, making it suitable for scalable vaccine or therapeutic development. By improving both the flexibility and functionality of IAV-ΔNS1 vectors, our work advances the utility of influenza A viruses as customizable tools for vaccine delivery, immune modulation, and therapeutic applications.