Influenza nucleoprotein DNA vaccination by a skin targeted, dry coated, densely packed microprojection array (Nanopatch) induces potent antibody and CD8(+) T cell responses.

DNA vaccines have many advantages such as thermostability and the ease and rapidity of manufacture; for example, in an influenza pandemic situation where rapid production of vaccine is essential. However, immunogenicity of DNA vaccines was shown to be poor in humans unless large doses of DNA are used. If a highly efficacious DNA vaccine delivery system could be identified, then DNA vaccines have the potential to displace protein vaccines. In this study, we show in a C57BL/6 mouse model, that the Nanopatch, a microprojection array of high density (>21,000 projections/cm(2)), could be used to deliver influenza nucleoprotein DNA vaccine to skin, to generate enhanced antigen specific antibody and CD8(+) T cell responses compared to the conventional intramuscular (IM) delivery by the needle and syringe. Antigen specific antibody was measured using ELISA assays of mice vaccinated with a DNA plasmid containing the nucleoprotein gene of influenza type A/WSN/33 (H1N1). Antigen specific CD8(+) T cell responses were measured ex-vivo in splenocytes of mice using IFN-γ ELISPOT assays. These results and our previous antibody and CD4(+) T cell results using the Nanopatch delivered HSV DNA vaccine indicate that the Nanopatch is an effective delivery system of general utility that could potentially be used in humans to increase the potency of the DNA vaccines.

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array.

The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads. Delivery of siRNA by such devices has been proven in principle, yet requires optimisation for clinical applications. Herein, we demonstrate the use of Nanopatch™ microprojection arrays to deliver liposome-encapsulated siRNA to overcome skin barrier, and in vivo siRNA delivery hurdles. This application provided effective silencing of CXCL1 expression induced by the co-delivery of Fluvax 2012® by microprojection array. Liposomes encapsulating siRNA were dry-coated onto microprojection arrays, and remained intact after elution from arrays in vitro. Microprojection arrays facilitated the delivery of fluorescently-labelled nucleic acids through murine ear stratum corneum to the epidermis and dermis, with diffusion from microprojections into adjacent skin evident within 30s. CXCL1 mRNA, induced by delivery of Fluvax by microprojection array, was reduced by 75% up to 20 h post-treatment by co-delivery of liposome-encapsulated CXCL1-specific siRNA, but not by arrays co-delivering liposome-encapsulated control siRNA. CXCL1 protein expression in explant cultures from skin treated with arrays bearing CXCL1 specific or control siRNA was similarly reduced. These results as a test case have many implications for gene silencing in skin and inflammation, with the benefit of targeted delivery using microprojection arrays to deliver liposome-encapsulated siRNA.

Dry-coated live viral vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific T cell responses in mice.

The disadvantages of needle-based immunisation motivate the development of simple, low cost, needle-free alternatives. Vaccine delivery to cutaneous environments rich in specialised antigen-presenting cells using microprojection patches has practical and immunological advantages over conventional needle delivery. Additionally, stable coating of vaccine onto microprojections removes logistical obstacles presented by the strict requirement for cold-chain storage and distribution of liquid vaccine, or lyophilised vaccine plus diluent. These attributes make these technologies particularly suitable for delivery of vaccines against diseases such as malaria, which exerts its worst effects in countries with poorly-resourced healthcare systems. Live viral vectors including adenoviruses and poxviruses encoding exogenous antigens have shown significant clinical promise as vaccines, due to their ability to generate high numbers of antigen-specific T cells. Here, the simian adenovirus serotype 63 and the poxvirus modified vaccinia Ankara--two vectors under evaluation for the delivery of malaria antigens to humans--were formulated for coating onto Nanopatch microprojections and applied to murine skin. Co-formulation with the stabilising disaccharides trehalose and sucrose protected virions during the dry-coating process. Transgene-specific CD8(+) T cell responses following Nanopatch delivery of both vectors were similar to intradermal injection controls after a single immunisation (despite a much lower delivered dose), though MVA boosting of pre-primed responses with Nanopatch was found to be less effective than the ID route. Importantly, disaccharide-stabilised ChAd63 could be stored for 10 weeks at 37°C with less than 1 log10 loss of viability, and retained single-dose immunogenicity after storage. These data support the further development of microprojection patches for the deployment of live vaccines in hot climates.

Rapid kinetics to peak serum antibodies is achieved following influenza vaccination by dry-coated densely packed microprojections to skin.

A rapid time to peak serum antibody response following vaccination is particularly important for influenza: the time window between the availability of appropriate antigen and the start of the seasonal epidemic is very short. In this paper, influenza vaccine was delivered to both the epidermis and dermis of mouse skin using densely packed microprojection arrays for vaccination. We found that, after vaccination, around 75% and 90% of the delivered influenza vaccine migrated away from the ear skin within just 2 days and 1 week - respectively. And the time to peak serum antibody response was as early as 2 weeks. This result matches the kinetics achieved by intramuscular injection of liquid vaccine to muscle. Thus, we demonstrate that skin delivery of small vaccine volumes discretely by thousands of densely packed microprojections neither induces delay in kinetics nor interferes with the long-lasting antibody response; compared to conventional intramuscular injection.

Nanopatch targeted delivery of both antigen and adjuvant to skin synergistically drives enhanced antibody responses.

Many vaccines make use of an adjuvant to achieve stronger immune responses. Alternatively, potent immune responses have also been generated by replacing the standard needle and syringe (which places vaccine into muscle) with devices that deliver vaccine antigen to the skin's abundant immune cell population. However it is not known if the co-delivery of antigen plus adjuvant directly to thousands of skin immune cells generates a synergistic improvement of immune responses. In this paper, we investigate this idea, by testing if Nanopatch delivery of vaccine - both the antigen and the adjuvant - enhances immunogenicity, compared to intramuscular injection. As a test-case, we selected a commercial influenza vaccine as the antigen (Fluvax 2008®) and the saponin Quil-A as the adjuvant. We found, after vaccinating mice, that anti-influenza IgG antibody and haemagglutinin inhibition assay titre response induced by the Nanopatch (with delivered dose of 6.5ng of vaccine and 1.4µg of Quil-A) were equivalent to that of the conventional intramuscular injection using needle and syringe (6000ng of vaccine injected without adjuvant). Furthermore, a similar level of antigen dose sparing (up to 900 fold) - with equivalent haemagglutinin inhibition assay titre responses - was also achieved by delivering both antigen and adjuvant (1.4µg of Quil-A) to skin (using Nanopatches) instead of muscle (intramuscular injection). Collectively, the unprecedented 900 fold antigen dose sparing demonstrates the synergistic improvement to vaccines by co-delivery of both antigen and adjuvant directly to skin immune cells. Successfully extending these findings to humans with a practical delivery device - like the Nanopatch - could have a huge impact on improving vaccines.

Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization.

Dry-coated microprojections can deliver vaccine to abundant antigen-presenting cells in the skin and induce efficient immune responses and the dry-coated vaccines are expected to be thermostable at elevated temperatures. In this paper, we show that we have dramatically improved our previously reported gas-jet drying coating method and greatly increased the delivery efficiency of coating from patch to skin to from 6.5% to 32.5%, by both varying the coating parameters and removing the patch edge. Combined with our previous dose sparing report of influenza vaccine delivery in a mouse model, the results show that we now achieve equivalent protective immune responses as intramuscular injection (with the needle and syringe), but with only 1/30th of the actual dose. We also show that influenza vaccine coated microprojection patches are stable for at least 6 months at 23°C, inducing comparable immunogenicity with freshly coated patches. The dry-coated microprojection patches thus have key and unique attributes in ultimately meeting the medical need in certain low-resource regions with low vaccine affordability and difficulty in maintaining "cold-chain" for vaccine storage and transport.