We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than 25 weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8+ DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly. © 2008 Elsevier Ltd. All rights reserved.