ORIGINAL ARTICLE C-di-GMP with influenza vaccine showed enhanced and shifted immune responses in microneedle vaccination in the skin Ju-Hyung Shin 1 & Ji-Ho Lee 2 & Seong Dong Jeong 1 & Jin-Yong Noh 2 & Hyo Won Lee 1 & Chang-Seon Song 2 & Yeu-Chun Kim 1 # Controlled Release Society 2020 Abstract A microneedle is a biomedical device which consists of multiple micron scale needles. It is widely used in various fields to deliver drugs and vaccines to the skin effectively. However, when considering improved vaccine efficacy in microneedle vaccination, it is important to find an appropriate adjuvant that is able to be used in transdermal delivery. Herein, we demonstrated the applicability of c-di-GMP, which is a stimulator of interferon genes (STING) agonist, as an adjuvant for influenza microneedle vaccination. Thus, 2 and 10 μg of GMP with the influenza vaccine were coated onto a microneedle, and then, BALB/c mice were immunized with the coated microneedle to investigate the immunogenicity and protection efficacy of the influenza microneedle vaccination. As a result, the adjuvant groups had an enhanced IgG response, IgG subtypes and HI titer compared to the vaccine only group. In addition to the humoral immunity, the use of an adjuvant has also been shown to improve the cellular immune response. In a challenge study, adjuvant groups had a 100% survival rate and rapid weight recovery. Taken together, this study confirms that GMP is an effective adjuvant for influenza microneedle vaccination. Keywords Microneedle . Adjuvant . Vaccination . STING agonist . Influenza Introduction Influenza vaccination is the most effective tools of preventing influenza outbreaks and pandemics, thereby reducing economical and human losses [1–5]. In order to further improve the efficacy of the influenza vaccination [4, 6], studies are constantly carried out in various fields from different perspectives [7]. For example, there are the development of vaccine antigens and adjuvants that can boost antigen immunity and improving of vaccine delivery methods [2]. Focusing on delivery methods, various tools, and delivery routes such as mucosal and transcutaneous injections have been studied to overcome the disadvan- tages of the traditional injection method using a hypoder- mic syringe into the muscle [8, 9]. Among skin and mu- cosal vaccination routes and injection devices, we intro- duced a microneedle, which is one of the skin vaccination tools, to improve the immune response to antigens and patient compliance [10]. This paper focused on a microneedle patch consisting of tiny needles less than 1 mm high targeting into the skin [10–12]. There are four types of microneedles, solid, coated, dissolving, and hollow microneedle, according to the shape and mechanism [10, 13, 14]. Coated microneedles, which are a patch with tips coated with the desired formulation, also have shown effective delivery of vaccines into the skin and have induced strong and long-term immune responses, as seen in other types of microneedles [15–18]. This study searched for a suitable adju- vant for a coated microneedle for an inactivated influenza vac- cine antigen to increase the immune response [19]. To further enhance the effect of this novel delivery method, we investigated adjuvants for use in this delivery method [20–23]. An adjuvant is an immune potentiator that improves the immunogenicity of an antigen when administered with an Ju-Hyung Shin and Ji-Ho Lee contributed equally to this work. * Chang-Seon Song songcs@konkuk.ac.kr * Yeu-Chun Kim dohnanyi@kaist.ac.kr 1 Department of Chemical and Biomolecular engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea 2 Avian Disease Laboratory, College of Veterinary Medicine, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea Drug Delivery and Translational Research https://doi.org/10.1007/s13346-020-00728-1