Skin Targeted Vaccinations Figure 3. Immune responses induced by influenza vaccine delivered by NanopatchTM and intramuscular injection. Nanopatches were coated with incremental and controlled doses of the vaccine FluvaxH2008 by the addition of different doses of FluvaxH in the same volume of methylcellulose. The doses indicated are the total hemagglutinin amounts of the three viral strains included in the vaccine. Seven microliter of the coating solution, containing FluvaxH and methylcellulose was applied on each NanopatchTM followed by drying with a stream of nitrogen gas. Delivered doses were determined by Coomassie Blue R-250 dye recovery. C57BL/6 mice were vaccinated once with one NanopatchTM on each inner ear lobe or by intramuscular injection with the indicated doses and were bled at 3 weeks and again at 2 months. (a,) Individual serum samples (3 weeks, blue circles; 2 months, Green squares) were assayed by ELISA on FluvaxH-coated wells and IgG titers for each individual animal are shown as a closed circle with the geometric mean titer (GMT) as a line. (b,) Sera (3 weeks, blue bars; 2 months, green bars) were assayed for hemagglutinin inhibition (HI) activity38 against each of the three component viruses included in the trivalent vaccine as indicated above the panels. Bars are the GMT and error bars are the standard error of the mean. doi:10.1371/journal.pone.0010266.g003 all unvaccinated mice were euthanized on day 7 post infection at the humane end-point after dramatic weight loss. Discussion In this study we demonstrated that the Nanopatches deliver influenza vaccine to directly contact thousands of skin APCs according to design, inducing functionally protective immune responses in mice comparable to the vaccine delivered intramus- cularly with the needle and syringe, but with less than 1/100th of the delivered dose. This result is an order of magnitude higher than has been previously reported with other delivery devices with only one vaccination, without using adjuvants (and thus, mitigating risk of adjuvant induced adverse reactions) [6]. This significant reduction in the amount of antigen needed to induce an immune response is most likely the result of directly targeting vaccine to a higher proportion of viable APCs in skin (e.g. achieved targeting of several thousands of Langerhans cells and dermal APCs per 0.32 cm2 of targeted skin) than current delivery methods. Although it is considered important to target antigen to APCs, it is not known how many cells should be targeted for effective or optimized vaccination. This is the case for both mice and humans. Therefore it follows that the thresholds for eliciting protective immune responses in both mice and humans is also not known. In this paper, we uniquely contribute to the body of knowledge by: (a). Using a probabilistically guided approach to directly target mice to more than 50% of both epidermal and dermal APCs (beneath the patch area). (b). Demonstrating by experiment in mice that antigen is directly deposited to thousands of epidermal and dermal APCs (the bodies of 1600 LC, up to 9000 when we consider the dendrites; to bodies of 680 dermal APC, up to 5000 Figure 4. Influenza challenge protection study using PR8 strain. Groups of five C57BL/6 mice were vaccinated with either PBS or different doses of a preparation of split virus vaccine comprising mouse-adapted influenza virus A/Puerto Rico 8/34. Mice were given the vaccine at the doses of 34 ng and 105 ng HA in the form of two Nanopatches to the ears, or with a dose of 6000 ng HA by IM injection with needle and syringe. Thirty six days after the vaccination, mice were challenged with 200 plaque forming units of virus (corresponding to approx. 4 times the lethal dose). The body weights of mice were recorded daily after the challenge. Mice were euthanized at a previously determined humane endpoint indicative of severe illness that would otherwise progress to death. doi:10.1371/journal.pone.0010266.g004 PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10266