performed using ABTS (2,29-azino-bis[3-ethylbenzthiazoline-6- sulfonic acid]) (Sigma cat. no. A-1888) as the substrate. The absorbance readings at 405 nm were measured against control wells containing no antiserum in the reaction. Each sample was individually analyzed. Hemagglutination-Inhibition Assay Sera were treated with receptor destroying enzyme (RDE II, Denka Seiken Co. Ltd.) prior to HI analysis to remove nonspecific inhibitors of agglutination. Samples were diluted 1in 5 in RDE and held at 37uC overnight. An equal volume of sodium citrate 1.6% (w/v) was then added and held at 56uC for 2 hr to neutralize RDE activity. The HI test was performed against each of the three purified influenza viruses present in the FluvaxH2008 formulation using chicken red blood cells by established methods [43] adapted to microtiter format. Viral challenge Mice (n = 5) were vaccinated with a preparation of mouse adapted influenza virus A/Puerto Rico/8/34 split virion [31]. Mice received 34 ng or 105 ng by NanopatchTM or 6000 ng by intramuscular injection. Unimmunized mice were used as a control. Thirty six days after the vaccination, mice were challenged with a lethal dose of 200 plaque forming units of the virus. Body weights and clinical signs of mice were recorded daily after challenge and, where necessary, mice were culled at a predetermined humane endpoint under guidelines approved by the University of Melbourne Animal Ethics Committee. Statistical analyses ELISA, HI titers and the data in the Fig 2 were analyzed by Mann-Whitney U test using GraphPad Prism 5 software (La Jolla, CA, USA). Supporting Information Figure S1 Figure S1 shows the normalized release of influenza vaccine into skin, following application of Nanopatches bearing References 1. Seielstad M (2000) Whiffs of selection. Nat Genet 26: 131–132. 2. Kupper TS, Fuhlbrigge RC (2004) Immune surveillance in the skin: Mechanisms and clinical consequences. Nat Rev Immunol 4: 211–222. 3. Kenney RT, Frech SA, Muenz LR, Villar CP, Glenn GM (2004) Dose sparing with intradermal injection of influenza vaccine. New Engl J Med 351: 2295–2301. 4. Van Damme P, Oosterhuis-Kafeja F, Van der Wielen M, Almagor Y, Sharon O, et al. (2009) Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults. Vaccine 27: 454–459. 5. Neumann G, Noda T, Kawaoka Y (2009) Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459: 931–939. 6. Petrovsky N, Aguilar JC (2004) Vaccine adjuvants: Current state and future trends. Immunol. Cell Biol 82: 488–496. 7. Palache B (2008) New vaccine approaches for seasonal and pandemic influenza. Vaccine 26: 6232–6236. 8. Laurent PE, Bonnet S, Alchas P, Regolini P, Mikszta JA, et al. (2007) Evaluation of the clinical performance of a new intradermal vaccine admini- stration technique and associated delivery system. Vaccine 25: 8833– 8842. 9. Zhu Q, Zarnitsyn VG, Ye L, Wen Z, Gao Y, et al. (2009) Immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge. Proc Natl Acad Sci USA 106: 7968–7973. 10. Koutsonanos DG, Martin MDP, Zarnitsyn VG, Sullivan SP, Compans RW, et al. (2009) Transdermal influenza immunization with vaccine-coated microneedle arrays. Plos One 4: e4773. 11. Matriano JA, Cormier M, Johnson J, Young WA, Buttery M, et al. (2002) Macroflux (R) microprojection array patch technology: A new and efficient approach for intracutaneous immunization. Pharm Res 19: 63–70. 12. Widera G, Johnson J, Kim L, Libiran L, Nyam K, et al. (2006) Effect of delivery parameters on immunization to ovalbumin following intracutaneous adminis- tration by a coated microneedle array patch system. Vaccine 24: 1653–1664. Skin Targeted Vaccinations dry-coated influenza vaccine to the inner lobe of the mouse ear for different times. The data shows that the released amount of influenza vaccine in the skin does not show statistical difference (p = 0.46) for Nanopatch application time of 2 and 5 minutes. For each application time (0.5, 2 and 5 minutes), a group of 6 coated patches were applied on 6 individual mouse ears for measurement of the released amount of coating in the skin. Video S1. This video shows part of a Nanopatch site in 3D. The Fluvax 2008H payload is red (Fluvax-Cy3) and the antigen presenting cells are shown in green (MHC class II). The image only contains the epidermis and the nuclei are shown in blue (Hoechst 33342). The skin was fixed in paraformaldehyde immediately after the patch was applied. Immunostaining for MHC class II immediately followed fixation. The Z stack video was generated by 3D rendering a z-stack in Imaris. The initial scene is the full image of Figure 2 (i)–(l). The movie then zooms in on a deposit site where the Fluvax co- localizes with an antigen presenting cell. Found at: doi:10.1371/journal.pone.0010266.s001 (0.25 MB TIF) Video S1 Multimedia File Video S1 Found at: doi:10.1371/journal.pone.0010266.s002 (6.02 MB AVI) Acknowledgments Nanopatches were fabricated at the Rutherford Appleton Laboratories, UK, by Derek Jenkins. Marion Brunck and Sally Yukiko are acknowledged for the technical assistance and Kerry Fagan, Chelsea Stewart, Trish Hitchcock and others at the Animal Facility in the AIBN at The University of Queensland for the animal care. We wish to thank Dr. Christopher Flaim for his suggestions. Author Contributions Conceived and designed the experiments: GJF XC TWP MSR IHF LEB MAK. Performed the experiments: GJF XC TWP MLC EJF. Analyzed the data: GJF XC TWP MLC EJF IHF LEB MAK. Contributed reagents/ materials/analysis tools: GJF XC TWP MLC EJF MSR IHF LEB MAK. Wrote the paper: GJF XC TWP MLC MSR LEB MAK. 13. Glenn GM, Kenney RT, Hammond SA, Ellingsworth LR (2003) Transcuta- neous immunization and immunostimulant strategies. Expert RevVaccines 2: 253–267. 14. Furth PA, Shamay A, Hennighausen L (1995) Gene transfer into mammalian cells by jet injection. Hybridoma 14: 149–152. 15. Kendall M, Mitchell T, Wrighton-Smith P (2004) Intradermal ballistic delivery of micro-particles into excised human skin for pharmaceutical applications. J Biomech 37: 1733–1741. 16. Merad M, Ginhoux F, Collin M (2008) Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nat Rev Immunol 8: 935–947. 17. Doukas AG, Flotte TJ (1996) Physical characteristics and biological effects of laser-induced stress waves. Ultrasound Med Biol 22: 151–164 (1996). 18. Han SW, Nakamura C, Obataya I, Nakamura N, Miyake J (2005) Gene expression using an ultrathin needle enabling accurate displacement and low invasiveness. Biochem Bioph Res Co 332: 633–639. 19. Palese P (2004) Influenza: old and new threats. Nat Med 10: S82–S87. 20. Poland GA (2006) Vaccines against avian influenza - A race against time. New Engl J Med 354: 1411–1413. 21. Mulholland WJ, Arbuthnott EAH, Bellhouse BJ, Cornhill JF, Austyn JM, et al. (2006) Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization. J Invest Dermatol 126: 1541–1548. 22. Koyama Y, Nagao S, Ohashi K, Takahashi H, Marunouchi T (1987) Sex- differences in the densities of epidermal Langerhans cells of the mouse. J Invest Dermatol 88: 541–544. 23. Yu RC, Abrams DC, Alaibac M, Chu AC (1994) Morphological and quantitative-analyses of normal epidermal Langerhans cells using confocal scanning laser microscopy. Brit J Dermatol 131: 843–848. 24. Lopez-Bravo M, Ardavin C (2008) In vivo induction of immune responses to pathogens by conventional dendritic cells. Immunity 29: 343–351. PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10266