Immunization in the Skin Local Response to Microneedle-Based Influenza 2012

Microneedle patches (MN) provide a novel method of vaccine delivery to the skin with the objective of targeting the large network of resident antigen-presenting cells to induce an efficient immune response. Our previous reports demonstrated that cutaneous delivery of inactivated influenza virus-coated MN to mice protects against lethal infection. Protection is correlated with sustained levels of anti-influenza virus serum antibodies, hemagglutination inhibition titers, and robust cellular responses that are often stronger than those generated by intramuscular vaccination. Here we dissect the early events occurring in murine skin after microneedle delivery of inactivated influenza virus. We demonstrate correlation of immunization against influenza virus with a local increase of cytokines important for recruitment of neutrophils, monocytes and dendritic cells at the site of immunization. We also observed prolonged antigen deposition, and migration of matured dendritic cells bearing influenza virus antigen from the skin. IMPORTANCE The immunological mechanisms by which MN vaccination confers protective immunity are not well understood. The present study provides a first analysis of the early immune events after microneedle-based vaccination. Received 12 January 2012 Accepted 2 February 2012 Published 1 March 2012 Citation del Pilar Martin M, et al. 2012. Local response to microneedle-based influenza immunization in the skin. mBio 3(2):e00012-12. doi:10.1128/mBio.00012-12. Editor Jack Bennink, National Institute of Allergy and Infectious Diseases Copyright © 2012 del Pilar Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Richard W. Compans, rcompan@emory.edu. Vaccination against infectious agents remains the most effective method of disease prevention and one of the most important health advances in history. Vaccines prevent or ameliorate infection based on two fundamental principles: the generation of a rapid and effective immune response, and long-lived specific immunity. Despite the success of vaccination, a number of challenging pathogens continue to elude complete protection by vaccines due to their rapid mutation capacity and/or the variety of subtypes that exist in nature. Due to the antigenic diversity observed among influenza virus strains and limited duration of the immune response to inactivated vaccines, annual vaccination is required (1). The influenza vaccine composition is determined every year based on the analysis of influenza surveillance activity reports, which help to estimate which strains of virus are expected to circulate in the human population (2). Efficacy of the inactivated influenza vaccine is correlated with seroconversion against the major influenza surface protein, the hemagglutinin (3, 4). Acceptable levels of hemagglutinin antibody titers are normally achieved by higher doses and/or multiple doses in individuals who have not been previously exposed to an influenza virus of the same subtype (5). Influenza vaccination could benefit from new vaccine formulations and novel vaccine delivery methods that provide an enhanced immune response. Alternate routes of vaccination to the conventional intramuscular delivery have been found to produce enhanced immune responses, and in humans, a nasally administered live attenuated influenza vaccine has also proven to be effective (6). However, this vaccine is approved only for healthy individuals 2 through 49 years of age, which excludes those at highest risk for developing influenza-related complications, such as infants, elderly persons, and immunocompromised individuals (7). One very attractive and promising alternative route of immunization is the skin. In 2011, the U.S. Food and Drug Administration (FDA) approved the first dermally administered influenza vaccine for human use in the United States (8, 9). The skin is not merely a physical barrier; a large body of work has demonstrated that the skin is also a complex and active immune site. Intradermal delivery provides access to the dense network of antigen-presenting cells, including macrophages, Langerhans cells, and dermal dendritic cells (DC) present in the dermis and epidermis, which are crucial in the initiation of the adaptive immune response and provide a favorable site for immunization (10, 11). Recent work from our laboratories and others has shown that vaccine delivery via the skin using metal microneedle patches (MN) coated with influenza antigens or dissolving microneedles with encapsulated inactivated influenza virus results in enhanced or equivalent protection compared to delivery via the conventional intramuscular or subcutaneous routes (12–16). Challenge studies in MN-immunized mice demonstrated complete protection against homosubtypic viruses. MN vaccination induced a broad spectrum of immune responses, including higher frequency of virus-specific CD4 and CD8 T cells, virus-specific antibodyRESEARCH ARTICLE March/April 2012 Volume 3 Issue 2 e00012-12 ® mbio.asm. org 1 m b.asm . rg on A ril 8, 2012 P ubished by m b.asm . rg D ow nladed fom secreting cells in the lung, and induction of a broader IgG isotype profile compared to conventional intramuscular delivery. These adaptive immune responses are the result of orchestrated events initiated by the innate immune system, which plays a fundamental role in sensing microbes and danger signals through pattern recognition receptors (PRR) (17). The plasticity of DC subsets and the PPR responses have been described as key initiating factors that ultimately determine the quality of the adaptive immune response (18). Therefore, we investigated the early events after MN vaccination in order to gain new insights into the innate immunological mechanisms involved in MN delivery. In this study we dissected the events that occur in the skin at early time points following influenza MN vaccination in mice. We analyzed the time course of antigen retention within the skin and the release of inflammatory cytokines mediated by MN insertion. Furthermore, we analyzed the egress of influenza antigen-bearing CD11c DC from the skin and their phenotype. These results provide evidence of robust local innate immune responses generated by MN delivery to the skin, and may provide insight into how immune responses may differ depending on the vaccination route.