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Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier to use and more cost-effective method for administration of vaccines when compared to the needle and syringe. Since MAPs deliver vaccine to the dermis and epidermis, a degree of local immune response at the site of application is expected. In a phase 1 clinical trial (ACTRN 12618000112268), the Vaxxas high-density MAP (HD-MAP) was used to deliver a monovalent, split inactivated influenza virus vaccine into the skin. HD-MAP immunisation led to significantly enhanced humoral responses on day 8, 22 and 61 compared with IM injection of a quadrivalent commercial seasonal influenza vaccine (Afluria Quadrivalent®). Here, the aim was to analyse cellular responses to HD-MAPs in the skin of trial subjects, using flow cytometry and immunohistochemistry. HD-MAPs were coated with a split inactivated influenza virus vaccine (A/Singapore/GP1908/2015 [H1N1]), to deliver 5 μg haemagglutinin (HA) per HD-MAP. Three HD-MAPs were applied to the volar forearm (FA) of five healthy volunteers (to achieve the required 15 μg HA dose), whilst five control subjects received three uncoated HD-MAPs (placebo). Local skin response was recorded for over 61 days and haemagglutination inhibition antibody titres (HAI) were assessed on days 1, 4, 8, 22, and 61. Skin biopsies were taken before (day 1), and three days after HD-MAP application (day 4) and analysed by flow-cytometry and immunohistochemistry to compare local immune subset infiltration. HD-MAP vaccination with 15 μg HA resulted in significant HAI antibody titres compared to the placebo group. Application of uncoated placebo HD-MAPs resulted in mild erythema and oedema in most subjects, that resolved by day 4 in 80% of subjects. Active, HA-coated HD-MAP application resulted in stronger erythema responses on day 4, which resolved between days 22-61. Overall, these erythema responses were accompanied by an influx of immune cells in all subjects. Increased cell infiltration of CD3+, CD4+, CD8+ T cells as well as myeloid CD11b+ CD11c+ and non-myeloid CD11b- dendritic cells were observed in all subjects, but more pronounced in active HD-MAP groups. In contrast, CD19+/CD20+ B cell counts remained unchanged. Key limitations include the use of an influenza vaccine, to which the subjects may have had previous exposure. Different results might have been obtained with HD-MAPs inducing a primary immune response. In conclusion, influenza vaccine administered to the forearm (FA) using the HD-MAP was well-tolerated and induced a mild to moderate skin response with lymphocytic infiltrate at the site of application., Competing Interests: Some of the authors were employed by commercial companies. Vaxxas Pty was the sole funder of the study, and the contributions of these authors were supported by Vaxxas, either as employees, consultants, or fee-for-service contracts. I have read the journal’s policy and the authors of this manuscript have the following competing interests: - AHF, ACID and KW are employees of Vaxxas Pty Ltd. - JWW, CDA, PT, JH and GJPF received consulting fees from Vaxxas Pty Ltd to support their contribution to this research. - JWW and MV are employees of the University of Queensland, which carried out some assay development on a contract basis paid for by Vaxxas Pty Ltd. - JDL, is an employee of Nucleus Network, which carried out the clinical trial (trial site) on a contract basis paid for by Vaxxas Pty Ltd. - This commercial affiliation does not alter Vaxxas’ adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data and/or materials.

Background: The Vaxxas high-density microarray patch (HD-MAP) consists of a high density of microprojections coated with vaccine for delivery into the skin. Microarray patches (MAPs) offer the possibility of improved vaccine thermostability as well as the potential to be safer, more acceptable, easier to use, and more cost-effective for the administration of vaccines than injection by needle and syringe (N&S). Here, we report a phase I trial using the Vaxxas HD-MAP to deliver a monovalent influenza vaccine that was to the best of our knowledge the first clinical trial to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs., Methods and Findings: HD-MAPs were coated with a monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/2015 H1N1 haemagglutinin (HA). Between February 2018 and March 2018, 60 healthy adults (age 18-35 years) in Melbourne, Australia were enrolled into part A of the study and vaccinated with either: HD-MAPs delivering 15 μg of A/Singapore/GP1908/2015 H1N1 HA antigen (A-Sing) to the volar forearm (FA); uncoated HD-MAPs; intramuscular (IM) injection of commercially available quadrivalent influenza vaccine (QIV) containing A/Singapore/GP1908/2015 H1N1 HA (15 μg/dose); or IM injection of H1N1 HA antigen (15 μg/dose). After 22 days' follow-up and assessment of the safety data, a further 150 healthy adults were enrolled and randomly assigned to 1 of 9 treatment groups. Participants (20 per group) were vaccinated with HD-MAPs delivering doses of 15, 10, 5, 2.5, or 0 μg of HA to the FA or 15 μg HA to the upper arm (UA), or IM injection of QIV. The primary objectives of the study were safety and tolerability. Secondary objectives were to assess the immunogenicity of the influenza vaccine delivered by HD-MAP. Primary and secondary objectives were assessed for up to 60 days post-vaccination. Clinical staff and participants were blind as to which HD-MAP treatment was administered and to administration of IM-QIV-15 or IM-A/Sing-15. All laboratory investigators were blind to treatment and participant allocation. Two further groups in part B (5 participants per group), not included in the main safety and immunological analysis, received HD-MAPs delivering 15 μg HA or uncoated HD-MAPs applied to the forearm. Biopsies were taken on days 1 and 4 for analysis of the cellular composition from the HD-MAP application sites. The vaccine coated onto HD-MAPs was antigenically stable when stored at 40°C for at least 12 months. HD-MAP vaccination was safe and well tolerated; any systemic or local adverse events (AEs) were mild or moderate. Observed systemic AEs were mostly headache or myalgia, and local AEs were application-site reactions, usually erythema. HD-MAP administration of 2.5 μg HA induced haemagglutination inhibition (HAI) and microneutralisation (MN) titres that were not significantly different to those induced by 15 μg HA injected IM (IM-QIV-15). HD-MAP delivery resulted in enhanced humoral responses compared with IM injection with higher HAI geometric mean titres (GMTs) at day 8 in the MAP-UA-15 (GMT 242.5, 95% CI 133.2-441.5), MAP-FA-15 (GMT 218.6, 95% CI 111.9-427.0), and MAP-FA-10 (GMT 437.1, 95% CI 254.3-751.3) groups compared with IM-QIV-15 (GMT 82.8, 95% CI 42.4-161.8), p = 0.02, p = 0.04, p < 0.001 for MAP-UA-15, MAP-FA-15, and MAP-FA-10, respectively. Higher titres were also observed at day 22 in the MAP-FA-10 (GMT 485.0, 95% CI 301.5-780.2, p = 0.001) and MAP-UA-15 (367.6, 95% CI 197.9-682.7, p = 0.02) groups compared with the IM-QIV-15 group (GMT 139.3, 95% CI 79.3-244.5). Results from a panel of exploratory immunoassays (antibody-dependent cellular cytotoxicity, CD4+ T-cell cytokine production, memory B cell (MBC) activation, and recognition of non-vaccine strains) indicated that, overall, Vaxxas HD-MAP delivery induced immune responses that were similar to, or higher than, those induced by IM injection of QIV. The small group sizes and use of a monovalent influenza vaccine were limitations of the study., Conclusions: Influenza vaccine coated onto the HD-MAP was stable stored at temperatures up to 40°C. Vaccination using the HD-MAP was safe and well tolerated and resulted in immune responses that were similar to or significantly enhanced compared with IM injection. Using the HD-MAP, a 2.5 μg dose (1/6 of the standard dose) induced HAI and MN titres similar to those induced by 15 μg HA injected IM., Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR.org.au), trial ID 108 ACTRN12618000112268/U1111-1207-3550., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: AHF, KW, ACID are paid employees of Vaxxas Pty Ltd. JWW, PT, GJPF and JH received consulting fees from Vaxxas Pty Ltd. JWW and MV are employees of the University of Queensland which carried out work for the study on a contract basis paid for by Vaxxas Pty Ltd. MP is an employee of 360Biolabs Pty Ltd which carried out work for the study on a contract basis paid for by Vaxxas Pty Ltd. JDL is an employee of Nucleus Network which carried out work for the study on a contract basis paid for by Vaxxas Pty Ltd. BF is an employee of Avance Clinical (formerly CPR Pharma Services) which carried out work for the study on a contract basis paid for by Vaxxas Pty Ltd. AW is an employee of Melbourne University which carried out work for the study on a contract basis paid for by Vaxxas Pty Ltd. AW has received travel expenses from Vaxxas Pty Ltd to attend data-review meetings.