The use of adjuvants with influenza vaccines has been shown to induce improved immune responses 
. However, these approaches have relied on the use of traditional needle-and-syringe delivery methods. Skin-based vaccination strategies using microneedle delivery systems are an attractive alternative and take advantage of the large population of innate immune cells including dermal dendritic cells and Langerhans cells. In addition, this vaccine delivery method has other advantages including a painless vaccination 
In the present study, we tested the hypothesis that microneedle delivery of the TLR7 and TLR3 ligands imiquimod and poly(I:C), respectively, to the skin with a licensed monovalent influenza H1N1 subunit vaccine will enhance the immune response in mice, compared to microneedle immunization with vaccine alone. We demonstrated that although the total IgG response was not significantly enhanced following microneedle immunization using influenza vaccine and adjuvant coated microneedles compared to vaccine alone, there were significant increases in the induction of IgG2a and functional antibodies blocking receptor binding using adjuvanted subunit vaccine and microneedle delivery.
CD4+ T cells play an important role in inducing class switching in B cells. In addition to induction of class switching to IgG2a, IFN-γ has also been shown to induce an anti-viral state, and upregulate MHC class I and II 
. Therefore we measured the expression of a Th1 (IFN-γ) and a Th2 (IL-4) cytokine in mice receiving adjuvanted microneedle vaccines. We observed that vaccination with influenza subunit vaccine and imiquimod resulted in a higher frequency of IFN-γ+ effector CD4+ T cells compared to mice immunized with subunit vaccine alone. This correlated with the induction of IgG2a in the sera of mice vaccinated with subunit vaccine and imiquimod.
Further investigation into the protective immune responses indicated that adjuvanted microneedle subunit vaccine improved resistance to lethal challenge compared to vaccine only. Notably, microneedle vaccination with subunit vaccine and imiquimod resulted in improved clearance of virus from lungs. Previous studies using the 1918 H1N1 virus and highly pathogenic avian H5N1 virus suggest that a strong induction of pro-inflammatory cytokines in the lungs results in immunopathology 
. We observed a reduction in pro-inflammatory cytokines in the lungs of mice that were immunized with subunit vaccine and imiquimod, which correlated with reduced body weight loss. These data suggest that microneedle vaccination with influenza subunit vaccine and imiquimod is capable of reducing immunopathology associated with infection.
It is interesting to note that the tissue expression pattern of TLRs varies greatly in mice. In murine skin, previous studies have demonstrated that isolated Langerhans cells express TLRs 2,4, and 9 mRNA 
. Fujita et al have demonstrated that murine Langerhans cells expressed TLR3 mRNA; however they exhibited low responsiveness to poly(I:C) 
. In our studies, delivering the TLR3 ligand, poly(I:C) using the coated microneedles did not enhance the immune response, in agreement with these findings. In addition, a recent study by Hasan et al 
suggests that responses to topical poly(I:C) can be inhibited by anti-microbial peptides. These antimicrobial peptides are upregulated in response to disruption of the skin barrier, suggesting that the lack of a response to poly(I:C) could be due in part to this response 
Conversely, TLR7 has not been shown to be expressed in the skin at steady state conditions 
. However, Suzuki et al have demonstrated that as a topically applied cream imiquimod induces migration of Langerhans cells from the skin and to the draining lymph nodes 
. Thus, the delivery of both vaccine antigen and imiquimod by coated microneedles allows for direct uptake of vaccine by Langerhans cells and TLR7-dependent enhancement of migration to draining lymph nodes.
Recent work by our lab investigated microneedle delivery of the A/Brisbane/59/2007 H1N1 subunit vaccine and compared immune responses to intramuscular vaccination 
. The results indicated that microneedle vaccination with 3 µg of A/Brisbane/59/2007 induced higher serum IgG2a levels compared to intramuscular vaccination at 4 weeks post-immunization. Furthermore, at 12 weeks post-immunization a higher frequency of IFN-γ secreting splenocytes was observed in mice vaccinated with coated microneedles compared to mice vaccinated intramuscularly suggesting a Th1 cytokine environment. Our present results with 1 µg of A/California/07/2009 antigen complement these results and suggest increased dose-sparing with similar body weight loss at 5×LD50
and improved survival after 20×LD50
challenge. The addition of MF59 has been shown to enhance immune responses to the California/07/2009 in adults receiving a single intramuscular vaccination with 3.75 µg of subunit vaccine 
. In the mouse model, MF59 adjuvanted A/California/07/2009 (0.5 or 1 µg) subunit vaccine was also more immunogenic compared to vaccine alone. 
. Although MF59 has been shown to be a very effective adjuvant, it is probably unsuitable to be coated onto solid microneedles due to its lipid content.
These results demonstrate the efficacy of vaccination using microneedles coated with influenza subunit vaccine and imiquimod. The imiquimod-adjuvanted vaccine induced greater functional antibody titers and enhanced cellular responses, inducing improved protective immune responses in mice. It is noteworthy that imiquimod is currently FDA-approved for human use when applied to the skin. In addition to TLR agonist adjuvants, other mediators of innate immunity including cytokines need to be investigated further. Taken together our results indicate that the use of adjuvants with coated-microneedle vaccines is a feasible approach for at enhancing the anti-viral immune response.