ABSTRACTThe influenza virus continues to pose a great threat to public health due to the frequent variations in RNA viruses. Vaccines targeting conserved epitopes, such as the extracellular domain of the transmembrane protein M2 (M2e), a nucleoprotein, and the stem region of hemagglutinin proteins, have been developed, but more efficient strategies, such as nanoparticle-based vaccines, are still urgently needed. However, the labor-intensivein vitropurification of nanoparticles is still necessary, which could hinder the application of nanoparticles in the veterinary field in the future. To overcome this limitation, we used regulated lysisSalmonellaas an oral vector with which to deliver three copies of M2e (3M2e-H1N1)-ferritin nanoparticlesin situand evaluated the immune response. Then, sequential immunization usingSalmonella-delivered nanoparticles followed by an intranasal boost with purified nanoparticles was performed to further improve the efficiency. Compared with 3M2e monomer administration,Salmonella-deliveredin situnanoparticles significantly increased the cellular immune response. Additionally, the results of sequential immunization showed that the intranasal boost with purified nanoparticles dramatically stimulated the activation of lung CD11b dendritic cells (DCs) and elevated the levels of effector memory T (TEM) cells in both spleen and lung tissues as well as those of CD4 and CD8 tissue-resident memory T (TRM) cells in the lungs. The increased production of mucosal IgG and IgA antibody titers was also observed, resulting in further improvements to protection against a virus challenge, compared with the pure oral immunization group.Salmonella-deliveredin situnanoparticles efficiently increased the cellular immune response, compared with the monomer, and sequential immunization further improved the systemic immune response, as shown by the activation of DCs, the production of TEMcells and TRMcells, and the mucosal immune response, thereby providing us with a novel strategy by which to apply nanoparticle-based vaccines in the future.IMPORTANCESalmonella-deliveredin situnanoparticle platforms may provide novel nanoparticle vaccines for oral administration, which would be beneficial for veterinary applications. The combination of administeringSalmonella-vectored, self-assembled nanoparticles and an intranasal boost with purified nanoparticles significantly increased the production of effector memory T cells and lung resident memory T cells, thereby providing partial protection against an influenza virus challenge. This novel strategy could open a novel avenue for the application of nanoparticle vaccines for veterinary purposes.