Influenza vaccines with enhanced immunogenicity are needed to improve protection, particularly for individuals at high risk of influenza-related complications. One of the approaches to achieve this goal is the evaluation of new vaccine delivery routes. Although the efficacy of intradermal vaccination has been proven to be successful for vaccines such as those against bacillus Calmette-Guérin, smallpox, rabies and, most recently, influenza, it is not widely practiced for other vaccines (25
). In recent studies, alternative delivery approaches designed to reach the dermal layer have demonstrated successful vaccine delivery and protective immune responses in animal models. In clinical studies, intradermal delivery of vaccine antigens, including influenza virus antigens, has shown promising results (26
). The main obstacle to the use of the intradermal delivery route is the lack of an appropriate delivery system that could circumvent the technically difficult Mantoux technique. Some examples of techniques that penetrate or disrupt the outermost layer of the skin, the stratum corneum, rely on the use of hypodermic microneedles, sandpaper, or electrical current.
Previously we demonstrated that MN immunization produces robust serum antibody responses and cellular responses capable of conferring protection against viral challenge at least as effectively as most conventional immunization routes (27
). Importantly, antibody titers remained elevated in the MN-immunized group even 6 months after vaccination and correlated with inhibition of viral replication and robust recall Th1 cellular responses after influenza virus challenge (28
). Therefore, the immune responses observed after a single MN dose not only are potent but also provide long-term immunity.
In the present study, we evaluated the innate immune responses in the skin that precede the development of the influenza virus-specific responses in order to investigate the mechanism of vaccine-induced immunity. Keratinocytes make up approximately 90% of the total cell population of the skin and play an important role in the innate immune response by secreting cytokines, chemokines, and antimicrobial peptides in response to foreign antigen (11
). Cross-talk between keratinocytes and Langerhans cells via these innate immune signals induces activation, maturation, and migration of antigen-presenting cells to draining lymph nodes. The importance of IL-1β and TNF-α in induction of Langerhans cell migration from the epidermis has been demonstrated in skin contact sensitization models and in experimental cutaneous Leishmania
). Our analysis of cytokine expression in the skin following insertion of antigen-coated microneedles indicates the upregulation of IL-1β, TNF-α, and MIP-1α and supports the current models of Langerhans cell migration. The induction of the chemotactic proteins MIP-1α, MIP-2, MCP-1, IP-10, G-CSF, and KC suggests that following antigen-coated microneedle vaccination, the production of IL-1β and TNF-α in the skin is reinforced by recruitment of neutrophils and macrophages to the site of vaccination (33
). Further work is needed to determine if the cytokine pattern observed is antigen specific, and such studies might offer a predictive signature that could serve to rapidly evaluate novel vaccine formulations.
Previous studies have demonstrated the migration of antigen-loaded Langerhans cells from mouse skin and subsequent homing to draining lymph nodes (34
). Such migration is important in priming naive lymphocytes and activation of the adaptive immune response. Here we have demonstrated the migration of activated and matured antigen-loaded CD11c+
DC from vaccinated skin with antigen-coated microneedles. These antigen-loaded CD11c+
DC also expressed low levels of CD8α and high levels of CD205, indicating that they were of skin origin (35
). In addition, these cells expressed high levels of MHC II and costimulatory molecules (CD86 and CD40), suggesting that they are capable of efficient activation of naive antigen-specific T lymphocytes. These results are consistent with previous studies indicating increased expression of CD80 and CD86 in Langerhans cells migrating from murine skin explants (36
Macroimaging of mice vaccinated with Qdot-labeled influenza virus indicates that antigen is deposited in the skin for up to 7 days (). This prolonged deposition of antigen suggests the formation of “antigen depots” at the sites of microneedle insertion. Antigen depot formation leads to prolonged antigen release, allowing efficient uptake by antigen-presenting cells (38
). Adjuvants such as aluminum hydroxide and incomplete Freund’s adjuvant are capable of forming antigen depots in addition to inducing the production of proinflammatory cytokines and granulocyte-recruiting chemokines, further serving their roles as immunostimulants (39
). It will be of interest to determine if the rate of release of vaccine from an antigen depot might depend on the large size of the inactivated virus antigen and the kinetics of antigen trafficking to the draining lymph nodes. In addition, we have shown that the local innate immune response is activated at the site of microneedle vaccination, resulting in the production of proinflammatory cytokines and chemokines. This early innate signaling induces activation and maturation of skin antigen-presenting cells, which have also captured antigen deposited by microneedle insertion. These cells migrate to draining lymph nodes, where they express CD40 and CD86 costimulatory molecules. Taken together, these results indicate that the efficiency of microneedle patch vaccination is controlled by the skin innate immune response and the migration of skin dendritic cell populations to the draining lymph nodes.
Using surface cell markers selected for murine DC characterization, CD205 and CD11c, we identified influenza virus-loaded DC and observed their migration from the skin, which suggests the capacity to travel to the lymph nodes. Our goal was to determine whether inactivated influenza virus used as a vaccine and delivered via MN was associated with skin dendritic cell maturation and to create parallels to research findings that have traditionally used fluorophores and ovalbumin as model antigens. This is of particular relevance in light of the differential T helper polarization observed at various skin immunization sites, correlating with site-specific DC distribution and dynamics (41
With the kinetics of antigen distribution and its dependence on the antigen and immunization route having been characterized, it will be of interest to investigate the different dendritic cell subpopulations involved in antigen presentation after inactivated-virus MN immunization. Moreover, the recent use of antibodies to target antigens to specific DC subsets has demonstrated the potential to enhance the magnitude of the adaptive immune response (42
). The changes in specific innate cells and the skin cytokine profile generated after vaccination are important parameters in characterizing the mechanisms involved in responses to skin immunization.