the Langerhans cells and dermal APCs previously measured [25]. We then considered the population of MHC class II cells are the Langerhans cells – with a density of 542617 cells/mm2 at the suprabasal location within the mouse ear (same strain and site) [22]. Thus, we derived from experiment that the bodies of approximately 1600 Langerhans cells were directly targeted by applying two of our prototype Nanopatches to each mouse. This result agrees approximately with our original theoretical expecta- tion of Nanopatches targeting 2000 Langerhans cell bodies; when we also take into account measurements showing that about 93% of the NanopatchTM projections penetrate the skin; then our theoretical targeted Langerhans cell body population becomes 1870 cells – even closer to the observed result in practice. We have shown that Nanopatches target vaccine to the desired skin APCs, in populations agreeing well with our original probability-based prediction used to guide the device design. When we also take into account the dendrites extending from these cell bodies, the number of APCs directly targeted by antigen is significantly greater. For example, for Langerhans cells, this could be greater than 9000 of the approximately 17000 resident Langerhans cells in skin under contact of both Nanopatches (0.32 cm2). Looking then deeper into the skin at the dermal APCs – within the same skin area under the Nanopatches – we determined by experiment that about 680 cell bodies were targeted (with further analysis of Fig 2i–l ) which is higher than the theoretical calculation of 450 (taking into account 93% of the projections penetrate the skin). When we theoretically also account for the dendrites, then we could have targeted antigen directly to as many as 5000 of the total of approximately 7360 dermal APCs. Therefore, we have confirmed by experiment that antigen was directly delivered to more than half of the APCs residing in both the epidermal and dermis – also agreeing very well with our probability-based analysis guiding our NanopatchTM design. NanopatchTM vaccination of mice yields equivalent total anti-influenza IgG responses as intramuscular injection, but with more than 100-fold less antigen delivered to the skin Mice were vaccinated once by applying Nanopatches coated with FluvaxH to both ears and a range of doses were tested across different experimental groups. After application, each Nano- patchTM was kept in place on the skin for 2 minutes, to give adequate time for the vaccine to dissolve and diffuse into the skin subsurface (Supporting Document Supplementaty Figure S1). Additional groups of mice were vaccinated by needle and syringe in the caudal thigh muscle. Following this single vaccination, all mice were bled at 3 weeks and also at 2 months. Serum antibodies (IgG) to vaccine antigens were assessed by ELISA [27] (Fig 3a). First, considering the 3 week bleed data (Fig 3a, upper panel) in needle and syringe vaccinated mice, reducing the dose by 10-fold gave a significantly lower response (p = 0.029, Mann-Whitney U test), and by a further 10-fold gave a weak but measureable response in only 3 of 4 animals. In contrast, antigen delivered to the skin using the NanopatchTM gave antibody responses even at the lowest measured delivered dose tested (6.5 ng), and with a dose of 34 ng showing no significant difference from the 6000 ng intramuscular dose (p = 0.11). This indicates that equivalent high antibody responses can be obtained with the NanopatchTM compared to intramuscular injection with more than 100-fold less antigen delivered to the skin. These responses persisted at high levels for at least 2 months (Fig 3a, lower panel). Skin Targeted Vaccinations More than a 100-fold reduction of delivered antigen dose was observed for functionally relevant antibody (HI) responses, for three strains, when the vaccine was administered using the NanopatchTM We then examined the functional relevance of the antibody produced in protection against the influenza virus. To do this, we tested the same sera against each of the three vaccine component strains of virus for hemagglutination-inhibitory (HI) activity (Fig. 3b). The HI activity assay is the most widely accepted ‘gold standard’ used as the surrogate for influenza protective effective- ness [28], [29]. First, we consider the H1N1 component of the Fluvax 2008H. We see, by three weeks after vaccination, that all the Nano- patchTM vaccinated groups – with delivered doses ranging from 538 ng down to 34 ng – generated equivalent or higher HI titers than 6000 ng intramuscularly vaccinated group. At the lowest NanopatchTM dose of just 6.5 ng, the HI titer was below responses generated by a 6000 ng intramuscular injection, but still greater than 600 ng intramuscularly vaccinated mice. Then, at two months after vaccination, the comparison between the Nano- patchTM and intramuscular groups yielded the same outcome. Second, turning to the H3N2 component of Fluvax 2008H, we observe that in all doses the NanopatchTM delivery (spanning from 538 ng right down to 6.5 ng) induced equivalent or higher HI titers compared to 6000 ng intramuscular injection, at three weeks following vaccination. Following this, at 2 months, we again see significant HI activity maintained in the NanopatchTM vaccinated mice compared to the intramuscular injected groups. With further examination of the H3N2 component, we see that while none of the intramuscular injections did achieve an HI titer of 1:40 (even with the highest dose) – which is the minimum level sufficient to correlate with protection in humans [30] ; the NanopatchTM- delivered vaccine yielded HI titers generally above this critical level. Third, considering the type B virus component of the Fluvax 2008H, we see NanopatchTM delivery of low doses of HA induced substantial HI activity above the critical value of 1:40 at 3 weeks, again indicative of protection at doses as low as 34 ng compared to 6000 ng with intramuscular injection. Furthermore, the antibody levels were maintained after 2 months above this critical level in all doses that were 34 ng or higher. Collectively for each of the three strains, more than a 100-fold reduction of delivered antigen dose was observed for HI responses when the vaccine was administered using the NanopatchTM – a similar magnitude of improvement as was observed from the IgG antibody responses in the ELISA. Delivering an influenza vaccine by NanopatchTM induces a protective immune response against viral challenge Although the HI assay is a widely accepted correlate for vaccination protection against influenza virus infection (discussed in many publications, with [28], [29] as two examples), we decided also to measure whether our NanopatchTM-delivered vaccine induced a protective response against influenza virus challenge. To carry out this part of the study, we could not use the commercial trivalent vaccine (FluvaxH 2008) because mice cannot be infected with these human strains of influenza. Therefore, as an alternative, we used a split virion vaccine preparation, based on the mouse- adapted A/Puerto Rico 8/34 strain [31] for use in the protection assays (described in Methods). We see in Fig. 4 that delivering the vaccine by NanopatchTM – even with delivered doses as low as 34 ng – induces a protective immune response, as mice exhibited no substantial weight loss or clinical signs of diseases. In contrast PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10266