1ECG). pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines. Vaccines are one of the most cost-effective medical interventions, with a profound benefit to mankind. Since their widespread introduction in the 20th century they are estimated to have prevented almost 6 million deaths per year, saved 386 million life years and saved more than 20 million childrens lives over the past 20 years1,2. Safety and tolerability concerns accompanying the use of vaccines that incorporate live-attenuated or killed microorganisms have led to the increasing use of Benzethonium Chloride vaccines composed of pathogen subunits. A drawback of this approach is that such subunit-based vaccines lack some of the inherent immunostimulatory properties of whole organism-based vaccines3. As such, they result in suboptimal humoral responses and low or no T cell responses, making multiple immunizations necessary to induce protective immunity3. Identification of effective adjuvants and alternative routes of immunization are important to overcome these challenges. Such advances have the potential to reduce the burden of re-vaccination and enhance vaccine efficacy, particularly in young, aged and immunocompromised populations. Currently used adjuvants consist of compounds that are co-injected with vaccine antigens, and include a variety of aluminum salts, immunostimulatory molecules, and emulsions containing oil and water4. While these adjuvant approaches can enhance the immune response to some vaccines, they are not universally effective. Moreover, because they are rarely studied in the absence of antigen, there are limited studies to define their mechanistic underpinnings5. In addition, the complexity Benzethonium Chloride and large molecular size of many adjuvants may hinder their incorporation into less invasive vaccine delivery strategies. The delivery of vaccine components through the skin via intradermal, subcutaneous, and intramuscular injection are the most common routes of immunization. Despite the well characterized importance of the epidermis in regulating cutaneous immune responses, its relevance to vaccination has received little attention because it is largely bypassed during injection-based vaccinations. It is likely that as less invasive approaches of vaccination, such as those utilizing microneedles or nanoparticles become more widespread, the need to fully understand the role of the epidermis in the context of vaccination will gain importance6,7,8. Likewise the identification and incorporation of Benzethonium Chloride topical agents that can act on cellular elements of the epidermis is an important approach to augment vaccine responses to cutaneous immunization9. The pressing need to develop approaches to enhance the response to influenza vaccination is widely recognized10. In addition, successful vaccination approaches using epicutaneous and microneedle delivery platforms have been thoroughly studied in models of influenza and there is evidence that perturbations of the epidermis can functionally influence the response to influenza vaccination11,12. In this study, we sought to determine if pharmacologic inhibition of signal transduction pathways could influence the response to influenza bHLHb27 vaccination by coupling the local application of an epidermal growth factor receptor (EGFR) inhibitor (EGFRI) with intradermal administration of influenza vaccine. We selected an inhibitor of the EGFR for the following reasons. Prior studies have shown that EGFRI therapy is associated with increased recruitment of immune cells such as dendritic cells and macrophages into the skin13. For our studies we selected an irreversible EGFRI (known as PD168393) because previous reports using murine models have shown that local application of this inhibitor to the skin enhances an antigen-specific cell-mediated immune response as well as blocks the immunosuppressive effects of ultraviolet radiation14,15. Consistent with their ability to influence cutaneous immune homeostasis, clinical trials using related irreversible EGFRIs such as dacomitinib and afatinib given systemically for the treatment of advanced cancer are associated with increased skin inflammation16,17. Thus, in both murine models and in clinical trials EGFRIs have been reported to influence immune cell trafficking and cutaneous immune homeostasis both of which are likely relevant to vaccination responses. Here, we demonstrate that topical application of a small molecule EGFR kinase inhibitor can be used to enhance the immune response to an intradermal influenza subunit vaccine. These data provide proof-of-concept evidence that kinase inhibitors delivered locally to the skin offer a novel approach to modulate cutaneous vaccine responses. Results Enhancement of humoral responses to influenza vaccine by the EGFRI PD168393 To determine the impact of a topical EGFRI on the response to influenza vaccination,.