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

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

doi:10.1016/j.virs.2026.09.009

October 2026

Citation: 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. Subcutaneous immunization with replication-competent live influenza virus confers durable protective immunity without respiratory pathology in mice .VIROLOGICA SINICA, 2026, 41(5) : 1184-1199. http://dx.doi.org/10.1016/j.virs.2026.09.009

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

Zhoulang Wang ^a,b ,
Chengxing Zhou ^b ,
Lixin Yin ^a ,
Xiaohong Liao ^b ,
Xiao Li ^c ,
Lei Li ^d,e ,
Zhichao Zhou ^f ,
Kangtian Li ^d ,
Meiyun Chen ^g ,
Dingbin Chen ^h ,
Minglei Liu ^b ,
Duo Xu ^b ,
Weibin She ^d ,
Qinhai Ma ^{c
,,} ,
Hui Cai ^{a
,,} ,
Rong Zhou ^{b,c
,,}

a. School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, 518107, China;
b. Guangzhou National Laboratory, Guangzhou International Bio Island, Guangzhou, 510005, China;
c. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510230, China;
d. Guangdong Nanshan Institute of Medical Innovation, Foshan, 528216, China;
e. School of Pharmacy, Tongji medical College, Huazhong University of Science and Technology, Wuhan, 430030, China;
f. Guangdong Polytechnic of Environmental Protection Engineering, Foshan, 528216, China;
g. School of Medicine, Southern University of Science and Technology, Shenzhen, 518055, China;
h. Institute of Medical Microbiology, Department of Immunology and Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China

Corresponding author: Qinhai Ma, 13268268214@163.com
Hui Cai, caihui5@mail.sysu.edu.cn
Rong Zhou, zhourong@vip.163.com
Received Date: 03 June 2026
Accepted Date: 14 September 2026
Available online: 21 September 2026

Abstract

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.
- Influenza virus
- , Live virus vaccine
- , Subcutaneous immunization
- , Durable immunity
- , Single-cell RNA sequencing
- , Protective immunity

Electronic Supplementary Material

10.1016j.virs.2026.09.009-ESM.docx
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