Zhoulang Wang, Chengxing Zhou, Lixin Yin, Xiaohong Liao, Xiao Li, Lei Li, Zhichao Zhou, Kangtian Li, Meiyun Chen, Dingbin Chen, Minglei Liu, Duo Xu, Weibin She, Qinhai Ma, Hui Cai, Rong Zhou
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.