It is possible that we underestimated the levels of IgA because of partial protein degradation, and we did not specifically measure NP-specific IgA in the bronchoalveolar lavages of these mice. Previous studies in IgA2/2 mice have suggested, however, that mucosal IgA, are not required, nor are they the primary cause for the superiority of the i.n. route of immunization. A recent study further suggested that protection from Moguisteine influenza challenge is determined by cooperativity between influenza virus-specific T cells, non-neutralizing anti-NP antibodies, and alveolar macrophages. Therefore, i.n. administration of IDLV could have the advantage of activating alveolar macrophages and other resident immune cell populations, in addition to stimulating antigen-specific cell-mediated and humoral responses. Additional studies are warranted to better characterize the immune cell subpopulations and effectors that are induced following immunization with IDLV-NP administered i.n., and to more fully elucidate the correlates of protection associated with this route. In this proof-of-concept study, we used influenza as a model system; however, developing an influenza vaccine was not the main purpose of this study, and our initial investigation of NP expressed from IDLV need not be considered as a final choice for an influenza vaccine. In our study, we could achieve full protection from influenza mortality in mice with 2 i.n. doses of IDLV-NP, but not with a single administration of IDLV-NP by either route. Alternative Ethynodiol diacetate strategies could improve the effectiveness of this approach. For instance, we could further diversify the immune response to our vaccine by adding another conserved influenza virus protein to IDLV-NP, such as M2. In the rAdV system, expression of NP and M2 together was more effective than either rAdV-NP or rAdV-M2 alone, and protection was achieved with a single dose of vaccine. Responses to IDLV may also be possibly be improved by heterologous prime/boost strategies using LVs with an envelope from a different serotype to further avoid the possibility of host anti-vector immunity.