This study tested the hypothesis that optimized microneedle patch formulations can stabilize trivalent subunit influenza vaccine during long-term storage outside the cold chain and when exposed to potential stresses found during manufacturing and storage. Formulations containing combinations of trehalose/sucrose, sucrose/arginine, and arginine/heptagluconate were successful at retaining most or all vaccine activity during storage at 25 °C for up to 24 months as determined by ELISA assay. The best formulation of microneedle patches contained arginine/heptagluconate, which showed no significant loss of vaccine activity during the study. To validate these in vitro findings, mice were immunized using trivalent influenza vaccine stored in microneedle patches for more than 1 year at 25 °C, which elicited antibody titers greater than or equal to fresh liquid vaccine delivered by intradermal injection, indicating the retention of immunogenicity during storage. Finally, influenza vaccine in microneedle patches lost no significant activity during exposure to 60 °C for 4 months, multiple freeze-thaw cycles, or electron beam irradiation. We conclude that optimally formulated microneedle patches can retain influenza vaccine activity during extended storage outside the cold chain and during other environmental stresses, which suggests the possibility of microneedle patch storage on pharmacy shelves without refrigeration.

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.

A novel hybrid delivery approach to achieve the delivery efficacy of injections, the safety, and patient compliance of the patch involved the designing and synthesis of the of rapidly dissolving polymer needles of micrometer dimensions for painless, self-administered delivery of the biomolecules, and especially for minimally invasive protein delivery. The researchers to achieve the goal encapsulated red-fluorescent bovine serum albumin within PVP polymer microneedles and delivered to procine skin. The fabrication developed is based on room-temperature in-situ polymerization, and also includes the details from the mechanical, encapsulation, and delivery properties of the resulting microneedles for the delivery of biomolecules to the skin. The development of microneedles has used the master structures made of a polymeric photoresist epoxy (SU-8) by a photolithography method, and then used three master structures to create reverse molds from polydimethylsiloxane (PDMS).

Virus-like particles (VLPs) have a number of features that make them attractive influenza vaccine candidates. Microneedle (MN) devices are being developed for the convenient and pain-free delivery of vaccines across the skin barrier layer. Whilst MN-based vaccines have demonstrated proof-of-concept in mice, it is vital to understand how MN targeting of VLPs to the skin epidermis affects activation and migration of Langerhans cells (LCs) in the real human skin environment. MNs coated with vaccine reproducibly penetrated freshly excised human skin, depositing 80% of the coating within 60 s of insertion. Human skin experiments showed that H1 (A/PR/8/34) and H5 (A/Viet Nam/1203/04) VLPs, delivered via MN, stimulated LCs resulting in changes in cell morphology and a reduction in cell number in epidermal sheets. LC response was significantly more pronounced in skin treated with H1 VLPs, compared with H5 VLPs. Our data provides strong evidence that MN-facilitated delivery of influenza VLP vaccines initiates a stimulatory response in LCs in human skin. The results support and validate animal data, suggesting that dendritic cells (DCs) targeted through deposition of the vaccine in skin generate immune response. The study also demonstrates the value of using human skin alongside animal studies for preclinical testing of intra-dermal (ID) vaccines.

Background. A major goal in influenza vaccine development is induction of serological memory and cellular responses to confer long-term protection and limit virus spread after infection. Here, we investigate induction of long-lived immunity against the 2009 H1N1 virus after skin vaccination. Methods. BALB/c mice received a single dose of 5 μg inactivated A/California/04/09 virus via coated metal microneedles (MN) applied to skin or via subcutaneous injection. Results. MN or subcutaneous vaccination elicited similar serum IgG and hemagglutination inhibition titers and 100% protection against lethal viral challenge 6 weeks after vaccination. Six months after vaccination, the subcutaneous group exhibited a 60% decrease in functional antibody titers and extensive lung inflammation after challenge with 10 × LD50 of homologous virus. In contrast, the MN group maintained high functional antibody titers and IFN-γ levels, inhibition of viral replication, and no signs of lung inflammation after challenge. MN vaccination conferred complete protection against lethal challenge, whereas subcutaneous vaccination induced only partial protection. These findings were further supported by high numbers of bone marrow plasma cells and spleen antibody-secreting cells detected in the MN group. Conclusions. A single skin vaccination with MN induced potent long-lived immunity and improved protection against the 2009 H1N1 influenza virus, compared with subcutaneous injection.

Very high vaccination coverage is required to eliminate measles, but achieving high coverage can be constrained by the logistical challenges associated with subcutaneous injection. To simplify logistics of vaccine delivery, a patch containing micron-scale polymeric needles was formulated to encapsulate the standard dose of measles vaccine (1000 TCID50) and the immunogenicity of the microneedle patch was compared with subcutaneous injection in rhesus macaques. The microneedle patch was administered without reconstitution with diluent, dissolved in skin within 10 minutes, and caused only mild, transient skin erythema. Both groups of rhesus macaques generated neutralizing antibody responses to measles that were consistent with protection and the neutralizing antibody titers were equivalent. In addition, the microneedle patches maintained an acceptable level of potency after storage at elevated temperature suggesting improved thermostability compared to standard lyophilized vaccine. In conclusion, a measles microneedle patch vaccine was immunogenic in non-human primates, and this approach offers a promising delivery method that could help increase vaccination coverage.

Purpose: To determine clearance kinetics and routes of clearance of molecules from the suprachoroidal space (SCS) of live New Zealand White rabbits. Methods: Suprachoroidal space collapse rate and pressure changes after microneedle injection into SCS were determined. Fluorescent fundus images were acquired to determine clearance rates of molecules ranging in size from 332 Da to 2 MDa. Microneedle injections of fluorescein were performed, and samples were taken from various sites over time to determine amount of fluorescein exiting the eye. Clearance transport was modeled theoretically and compared with experimental data. Results: After injection, pressures in SCS and vitreous humor spiked and returned to baseline within 20 minutes; there was no difference between these two pressures. Suprachoroidal space collapse occurred within 40 minutes. One hour after fluorescein injection, 46% of fluorescein was still present in the eye, 15% had transported across sclera, 6% had been cleared by choroidal vasculature, and 4% had exited via leakage pathways. Characteristic clearance time increased in proportion with molecular radius, but total clearance of 2 MDa FITC-dextran was significantly slower (21 days) than smaller molecules. These data generally agreed with predictions from a theoretical model of molecular transport. Conclusions: Guided by experimental data in the context of model predictions, molecular clearance from SCS occurred in three regimes: (1) on a time scale of approximately 10 minutes, fluid and molecules exited SCS by diffusion into sclera and choroid, and by pressure-driven reflux via transscleral leakage sites; (2) in approximately 1 hour, molecules cleared from choroid by blood flow; and (3) in 1 to 10 hours, molecules cleared from sclera by diffusion and convection.

Purpose To determine the effect of injection volume and formulation of a microneedle injection into the suprachoroidal space (SCS) on SCS thickness and closure kinetics. Methods Microneedle injections containing 25 to 150 μL Hanks' balanced salt solution (HBSS) were performed in the rabbit SCS ex vivo. Distribution of SCS thickness was measured by ultrasonography and three-dimensional (3D) cryo-reconstruction. Microneedle injections were performed in the rabbit SCS in vivo using HBSS, Discovisc, and 1% to 5% carboxymethyl cellulose (CMC) in HBSS. Ultrasonography was used to track SCS thickness over time. Results Increasing HBSS injection volume increased the area of expanded SCS, but did not increase SCS thickness ex vivo. With SCS injections in vivo, the SCS initially expanded to thicknesses of 0.43 ± 0.06 mm with HBSS, 1.5 ± 0.4 mm with Discovisc, and 0.69 to 2.1 mm with 1% to 5% CMC. After injection with HBSS, Discovisc, and 1% CMC solution, the SCS collapsed to baseline with time constants of 19 minutes, 6 hours, and 2.4 days, respectively. In contrast, injections with 3% to 5% CMC solution resulted in SCS expansion to 2.3 to 2.8 mm over the course of 2.8 to 9.1 hours, after which the SCS collapsed to baseline with time constants of 4.5 to 9.2 days. Conclusions With low-viscosity formulations, SCS expands to a thickness that remains roughly constant, independent of the volume of fluid injected. Increasing injection fluid viscosity significantly increased SCS thickness. Expansion of the SCS is hypothesized to be controlled by a balance between the viscous forces of the liquid formulation and the resistive biomechanical forces of the tissue.

Measles vaccination programs would benefit from delivery methods that decrease cost, simplify logistics, and increase safety. Conventional subcutaneous injection is limited by the need for skilled healthcare professionals to reconstitute and administer injections, and by the need for safe needle handling and disposal to reduce the risk of disease transmission through needle re-use and needlestick injury. Microneedles are micron-scale, solid needles coated with a dry formulation of vaccine that dissolves in the skin within minutes after patch application. By avoiding the use of hypodermic needles, vaccination using a microneedle patch could be carried out by minimally trained personnel with reduced risk of blood-borne disease transmission. The goal of this study was to evaluate measles vaccination using a microneedle patch to address some of the limitations of subcutaneous injection. Viability of vaccine virus dried onto a microneedle patch was stabilized by incorporation of the sugar, trehalose, and loss of viral titer was less than 1log 10 (TCID 50 ) after storage for at least 30 days at room temperature. Microneedle patches were then used to immunize cotton rats with the Edmonston-Zagreb measles vaccine strain. Vaccination using microneedles at doses equaling the standard human dose or one-fifth the human dose generated neutralizing antibody levels equivalent to those of a subcutaneous immunization at the same dose. These results show that measles vaccine can be stabilized on microneedles and that vaccine efficiently reconstitutes in vivo to generate a neutralizing antibody response equivalent to that generated by subcutaneous injection.

It is a high priority to develop a simple and effective delivery method for a cross-protective influenza vaccine. We investigated skin immunization by microneedle (MN) patch with human influenza split vaccine and virus-like particles containing heterologous M2 extracellular (M2e) domains (M2e5x virus-like particles (VLP)) as a cross-protective influenza vaccine candidate. Co-delivery of influenza split vaccine and M2e5x VLP to the skin by MN patch was found to confer effective protection against heterosubtypic influenza virus by preventing weight loss and reducing lung viral loads. Compared to intramuscular immunization, MN-based delivery of combined split vaccine and M2e5x VLPs shaped cellular immune responses toward T helper type 1 responses increasing IgG2a isotype antibodies as well as IFN-γ producing cells in mucosal and systemic sites. This study provides evidence that potential immunological and logistic benefits of M2e5x VLP with human influenza split vaccine delivered by MN patch can be used to develop an easy-to-administer cross-protective influenza vaccine.