Transcriptional effects of influenza virus M1 and NP gene expression in DF-1 cells and their immunogenicity in cognate B21 chickens

Although the influenza A virus (IAV) matrix protein 1 (M1) and nucleoprotein (NP) are attractive targets for cross-protective vaccines, their independent effects on host cell transcriptomes and subsequent immunogenicity in histocompatible hosts remain largely unexplored. Here, we utilized a previously established histocompatible DF-1 cell and a cognate B21 chicken (DF-1/B21) platform to characterize these interactions. Stable DF-1 cell lines expressing M1 or NP were analyzed via transcriptomic profiling and subsequently used as single-antigen vaccines. Transcriptional analysis revealed distinct host signatures: M1 expression was primarily associated with alterations in membrane-associated and cytoskeletal pathways. In contrast, NP expression was linked to the broad downregulation of DNA replication and cell cycle regulators, actin network reorganization, and the modulation of early innate signaling without inducing a full interferon response. Functional assays corroborated these findings, showing that despite higher viral susceptibility in M1-expressing cells, NP expression established a potent antiviral state characterized by reduced viral replication and an increase in pro-inflammatory (TNF-α, IL-6) and immune-recruiting (IL-15, IL-12p40) cytokines in vitro. NP, but not M1, was also spontaneously released into culture supernatants. In vivo, both vaccines elicited measurable humoral responses and reduced viral shedding upon H9N2 challenge. Furthermore, distinct shifts in peripheral CD8⁺/CD4⁺ ratios post-challenge indicated that both antigens effectively modulated cellular immunity. Collectively, these findings validate the DF-1/B21 system as a versatile platform for dissecting host–pathogen interactions. By effectively bridging cellular transcriptomics with systemic immunity, this histocompatible model offers a valuable framework for the broad evaluation of avian immunogens and the rational design of future poultry vaccines.