Potent Immunity to Low Doses of Influenza Vaccine by Probabilistic Guided Micro-Targeted Skin Delivery in a Mouse Model Germain J. P. Fernando1., Xianfeng Chen1., Tarl W. Prow1, Michael L. Crichton1, Emily J. Fairmaid4, Michael S. Roberts2, Ian H. Frazer3, Lorena E. Brown 4 , Mark A. F. Kendall1,3* 1 Delivery of Drugs and Genes Group (D2G2), Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia, 2 Therapeutics Research Unit, School of Medicine, The University of Queensland, Brisbane, Queensland, Australia, 3 Diamantina Institute for Cancer, Immunology & Metabolic Medicine, The University of Queensland, Brisbane, Queensland, Australia, 4 Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria, Australia Abstract Background: Over 14 million people die each year from infectious diseases despite extensive vaccine use [1]. The needle and syringe—first invented in 1853—is still the primary delivery device, injecting liquid vaccine into muscle. Vaccines could be far more effective if they were precisely delivered into the narrow layer just beneath the skin surface that contains a much higher density of potent antigen-presenting cells (APCs) essential to generate a protective immune response. We hypothesized that successful vaccination could be achieved this way with far lower antigen doses than required by the needle and syringe. Methodology/Principal Findings: To meet this objective, using a probability-based theoretical analysis for targeting skin APCs, we designed the NanopatchTM, which contains an array of densely packed projections (21025/cm2) invisible to the human eye (110 mm in length, tapering to tips with a sharpness of ,1000 nm), that are dry-coated with vaccine and applied to the skin for two minutes. Here we show that the Nanopatches deliver a seasonal influenza vaccine (FluvaxH 2008) to directly contact thousands of APCs, in excellent agreement with theoretical prediction. By physically targeting vaccine directly to these cells we induced protective levels of functional antibody responses in mice and also protection against an influenza virus challenge that are comparable to the vaccine delivered intramuscularly with the needle and syringe—but with less than 1/100th of the delivered antigen. Conclusions/Significance: Our results represent a marked improvement—an order of magnitude greater than reported by others—for injected doses administered by other delivery methods, without reliance on an added adjuvant, and with only a single vaccination. This study provides a proven mathematical/engineering delivery device template for extension into human studies—and we speculate that successful translation of these findings into humans could uniquely assist with problems of vaccine shortages and distribution—together with alleviating fear of the needle and the need for trained practitioners to administer vaccine, e.g., during an influenza pandemic. Citation: Fernando GJP, Chen X, Prow TW, Crichton ML, Fairmaid EJ, et al. (2010) Potent Immunity to Low Doses of Influenza Vaccine by Probabilistic Guided Micro-Targeted Skin Delivery in a Mouse Model. PLoS ONE 5(4): e10266. doi:10.1371/journal.pone.0010266 Editor: Anthony R. Fooks, Veterinary Laboratories Agency, United Kingdom Received December 21, 2009; Accepted March 28, 2010; Published April 21, 2010 Copyright: 2010 Fernando et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Health and Medical Research Council (Australia) Grants ID# 569726 (MAFK, GJPF, LEB), ID# 456150 (MAFK), Australian Research Council Grant ID# DP077464 (MAFK, MSR) and the Queensland Smart State Scheme funded this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.kendall@uq.edu.au . These authors contributed equally to this work. Introduction Vaccines can be more effective if they are delivered into the narrow layer just beneath the skin surface that contains a high density of antigen presenting cells (APCs) required to generate an immune response [2–4], rather than into the muscle where such cells present at a much lower density (Fig 1). One key focus for improvement is in achieving successful vaccination using the lowest dose possible. This is particularly important in the context of a rapidly emerging disease, such as pandemic influenza, where existing vaccination production methods are slow to meet the demand for population protection [5]. Some success in tackling this problem has been achieved by supplementing the vaccine with an adjuvant – although in many cases with an increase of adverse reactions [6]. Alternatively, targeting of vaccines directly to large populations of skin immune cells holds great potential in achieving protection, with significant dose sparing and improved tolerability profiles. So far, however, only limited dose sparing gains have been achieved: delivery approaches vaccinating with conventional antigens have yielded functional disease protection, but with dose sparing gains of one order of magnitude compared to intramus- cular injections without the use of an adjuvant. Most studies make PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10266