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

Influenza A viruses (IAVs) lacking the nonstructural protein 1 (NS1) gene (IAV-ΔNS1) are attenuated both in vitro and in vivo, yet retain replicative capacity in interferon signaling-deficient systems. To date, several efforts have been pursued to expand its functionality by incorporating transgenes in lieu of the NS1 gene. However, such vectors require complex segment designs, retain residual NS1 peptide stretches, or display suboptimal cytokine secretion, largely due to the constraints related to the partial nuclear export protein (NEP) open reading frame (ORF) upstream of the transgene integration site. Here, we describe a novel segment 8 design that can accommodate a transgene, where the complete NEP ORF is positioned downstream of the splicing acceptor site, thereby mitigating previous genetic constraints. Using this strategy, we successfully generated a recombinant IAV-ΔNS1 virus carrying the human interleukin 2 (IL-2) gene, inducing robust secretion of bioactive IL-2 upon infection. The vector demonstrated high replicative capacity, achieving titers comparable to wild-type viruses in MDCK cells expressing NS1 protein, and exhibited genetic stability over 10 successive passages. Importantly, infection induced notable viral antigen expression and high levels of bioactive IL-2 secretion in interferon-competent systems, such as MDCK, A549, and HEK293T cells in vitro and in mice upon pulmonary delivery in vivo. 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.

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.