Subcutaneous immunization with replication-competent live influenza virus confers durable protective immunity without respiratory pathology in mice.

Current influenza vaccination strategies are constrained by the trade-off between the safety of inactivated platforms and the immunogenic potency of live attenuated vaccines. Here, we provide a comprehensive demonstration that subcutaneous (SC) immunization with a non-attenuated, replication-competent live influenza A virus (LIV) decouples immunogenicity from pathogenicity. Using a murine model, we show that a single high-dose SC immunization of A/Puerto Rico/8/34 (H1N1) elicited sterile pulmonary immunity against lethal challenge, significantly outperforming intramuscular delivery and inactivated vaccines. Biodistribution analysis confirms viral replication is transient (< 7 days) and strictly restricted to the injection site and draining lymph nodes, with no systemic dissemination to the lungs or central nervous system. Mechanistically, single-cell transcriptomics reveal that SC delivery triggers a localized type I interferon "firewall" that transitions into a robust germinal center (GC) reaction, fostering extensive plasma cell differentiation and superior memory B cell formation compared to phosphate-buffered saline (PBS)-treated controls. This regimen conferred 100% protection at 277 days post-immunization. Together, these findings establish peripheral LIV administration as a high-potency paradigm for next-generation rapid-response vaccines.