Redirecting NEU (neuraminidase) antigen to autophagosomes confers enhanced cross-reactive T-cell immunity against heterosubtypic influenza virus infection

  • Autophagy. 2026 Jun;22(6):1186-1202. doi: 10.1080/15548627.2026.2629285.
Zirong Han  1  2  3 ,  Weiqi Pan  4 ,  Wenlong Lai  5 ,  Mingting Cui  1  2 ,  Ruiting Li  1  2 ,  Lisha Deng  1  2 ,  Yu Gao  5 ,  Silk J Shi  6 ,  Jianhui Gan  5 ,  Bruce T Lahn  6  7 ,  Yao-Qing Chen  1  2  3 ,  Yuelong Shu  1  2  8 ,  Caijun Sun  1  2  3
Affiliations
  • 1. School of Public Health (Shenzhen), Sun Yat-Sen University, Shenzhen, China.
  • 2. Shenzhen Key Laboratory of Pathogenic Microbes and Biosafety, Shenzhen Campus of Sun Yat-Sen University, Shenzhen, China.
  • 3. Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, China.
  • 4. State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
  • 5. Shenzhen Kangtai Biological Products Co., Ltd, Shenzhen, China.
  • 6. VectorBuilder, Chicago, IL, USA.
  • 7. Future Institute of Gene Delivery Research, Guangzhou, China.
  • 8. Key Laboratory of Pathogen Infection Prevention and Control (MOE), State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
PMID: 41655133 DOI: 10.1080/15548627.2026.2629285
Abstract

NEU (neuraminidase) is a potential cross-reactive antigen for developing broadly protective influenza vaccine, but has suboptimal immunogenicity. We here report that, when NEU antigen was redirected into phagophores, and subsequently autophagosomes, by fusing with MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; NEU-LC3B), it could efficiently activate the autophagosome-lysosome-major histocompatibility complex class II (MHC II) compartment pathway, and thus substantially improve the magnitude, breadth, and polyfunctionality of NEU-specific T cell immunity in mice. Remarkably, we identified several novel NEU-specific T-cell epitopes in response to NEU-LC3B-based immunization. Furthermore, mice immunized with NEU-based constructs were challenged with homologous A/CA/04/09 (H1N1), heterologous within-subtype strain A/Puerto Rico/8/1934 (PR8) (H1N1), and heterosubtypic A/Aichi/2/1968 (H3N2) virus, and the results demonstrated that NEU-LC3B-based vaccine provided a sterilizing immunity to homologous strains and cross-protection against antigenically distinct heterosubtypic challenge. In addition, cell depletion experiment demonstrated that T-cell-mediated immunity contributed to the NEU-LC3B-mediated immune protection. Collectively, this engineered NEU antigen with optimal immunogenicity represents a promising strategy for developing broadly protective influenza vaccines.Abbreviations: BSA, bovine serum albumin; CQ, chloroquine; ELISpot, enzyme-linked immunosorbent spot; HA, hemagglutinin; ICS, intracellular cytokine staining; IFNG/IFN-γ, interferon gamma; LD50, Median Lethal Doses; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NEU, neuraminidase; NP, nucleoprotein; PBS, phosphate-buffered saline; RAPA, rapamycin; SFCs, spot-forming cells; SV, split vaccine; TCID50, median tissue culture infectious dose; VSV, vesicular stomatitis virus; VSVΔG, VSV vector with the deletion of the G gene.

Keywords
Autophagy; T-cell epitopes; T-cell immunity; broadly protective influenza vaccine; neuraminidase.
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