Investigation of cell death caused by the Nanopatch™ and its role in vaccine delivery as a physical immune enhancer

Since the introduction of vaccines, millions of lives have been saved. Yet, infectious diseases continue to contribute to deaths in underdeveloped countries, with over 2 million children dying per year from preventable diseases such as diphtheria, pertussis and tetanus. Despite the advantages of vaccines, there are also some risks and disadvantages associated with vaccines and their delivery such as the need for medical personnel for administration, refrigeration of the vaccine, disease transmission associated with needles and potential adverse side effects due to adjuvants. While intramuscular and subcutaneous injections are most frequently used for vaccine administration, these sites are poor in antigen-presenting cells. The skin, the largest immunological organ, by contrast contains a dense network of antigen-presenting cells, important in the uptake of antigen and presentation to naive T-cells, and has received increased interest as a target site for vaccine delivery. Intradermal immunisations have achieved significant dose-sparing over conventional immunisation routes. However, using existing needle designs, intradermal injections can be difficult to administer into the thin dermal layer due to the proportionally large scale of the needle. This has led to the development of alternative skin-based delivery methods, such as microneedle arrays. Here, the Nanopatch™ is an array containing 3364 microprojections of ~110 µm in length, which was used to deliver the vaccine into the viable epidermis and dermis of the skin. With an unprecedented >100-fold dose-sparing reported by the Nanopatch™ in comparison to conventional immunisation strategies with an influenza split virion vaccine, the mechanisms behind this skin-mediated ‘adjuvant’ effect are not clear. Danger signals released by stressed, damaged or dead cells have resulted in enhanced immune responses in a similar manner to chemical adjuvants. Co-administrations of dead cells with vaccines have led to enhanced immune responses. While the adjuvanting effects of dead cells have been well established, the local or wide-spread level of cell damage inflicted by vaccine delivery devices have not been studied in the context of immunogenicity. Thus, the underlying hypothesis of this doctoral thesis was that Nanopatch™ projections penetrating the skin could convey a ‘physical immune enhancer’ effect similar to an adjuvant as a direct result of local tissue damage/ cell death by the device itself, responsible for the enhanced immunogenicity relative to needle-based routes. The extent of cell death after Nanopatch™ application and intradermal injection was investigated, characterised and quantified by Confocal/ Multiphoton microscopy, Western Blot and flow cytometry. A distinct pattern of dead cells was obtained with significantly higher levels (~65-fold) of cell death in murine ear skin following Nanopatch™ treatment than intradermal injection. Measured skin cell death was tuneable by changing projection density but not shapes, and was associated with modelled stresses of ≥1 MPa. Immunogenicity studies demonstrated consistently and statistically significantly higher anti-IgG endpoint titres (up to 50-fold higher) in Nanopatch™ than intradermally injected groups after delivery of a split virion influenza vaccine, with a 10-fold dose-sparing effect. Importantly, co-localisation of delivered antigen with both, live and dead cells was necessary for immunogenicity enhancement. Overall, higher inflammatory responses and longer-lasting antigen depot formation were associated with Nanopatch™ immunisations but not with intradermal injections. The findings indicated a correlation between co-localisation of antigen cell death caused by the Nanopatch™ and enhanced immunogenicity with split virion influenza vaccine as model antigen. Collectively, the data presented within this doctoral thesis propose a ‘physical immune enhancer’ effect by the Nanopatch™ that increases immune responses to vaccines. Elucidating the key differences between intradermal and microneedles or microprojections such as the Nanopatch™ may contribute to understand the different mechanisms between skin immunisation routes. This work contributes extensively to the field of skin-based immunisations by providing insights on how device-induced cell death and the resulting inflammation may affect immunogenicity. This physical immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in skin-based vaccine delivery.