Enhancement of cellular immunity following needle-free vaccination of mice with SARS-CoV-2 spike protein.

The COVID-19 pandemic has highlighted the need for vaccines capable of providing rapid and robust protection. One way to improve vaccine efficacy is delivery via microarray patches, such as the Vaxxas high-density microarray patch (HD-MAP). We have previously demonstrated that delivery of a SARS-CoV-2 protein vaccine candidate, HexaPro, via the HD-MAP induces potent humoral immune responses. Here, we investigate the cellular responses induced by HexaPro HD-MAP vaccination. We found that delivery via the HD-MAP induces a type one biassed cellular response of much greater magnitude as compared to standard intramuscular immunization.

Dermal Delivery of a SARS-CoV-2 Subunit Vaccine Induces Immunogenicity against Variants of Concern.

The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.

Developing a Stabilizing Formulation of a Live Chimeric Dengue Virus Vaccine Dry Coated on a High-Density Microarray Patch.

Alternative delivery systems such as the high-density microarray patch (HD-MAP) are being widely explored due to the variety of benefits they offer over traditional vaccine delivery methods. As vaccines are dry coated onto the HD-MAP, there is a need to ensure the stability of the vaccine in a solid state upon dry down. Other challenges faced are the structural stability during storage as a dried vaccine and during reconstitution upon application into the skin. Using a novel live chimeric virus vaccine candidate, BinJ/DENV2-prME, we explored a panel of pharmaceutical excipients to mitigate vaccine loss during the drying and storage process. This screening identified human serum albumin (HSA) as the lead stabilizing excipient. When bDENV2-coated HD-MAPs were stored at 4 °C for a month, we found complete retention of vaccine potency as assessed by the generation of potent virus-neutralizing antibody responses in mice. We also demonstrated that HD-MAP wear time did not influence vaccine deposition into the skin or the corresponding immunological outcomes. The final candidate formulation with HSA maintained ~100% percentage recovery after 6 months of storage at 4 °C.

Complete protection by a single-dose skin patch-delivered SARS-CoV-2 spike vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people and resulted in more than 3.3 million deaths, and despite the availability of multiple vaccines, the world still faces many challenges with their rollout. Here, we use the high-density microarray patch (HD-MAP) to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show that the vaccine is thermostable on the patches, with patch delivery enhancing both cellular and antibody immune responses. Elicited antibodies potently neutralize clinically relevant isolates including the Alpha and Beta variants. Last, a single dose of HD-MAP­delivered spike provided complete protection from a lethal virus challenge in an ACE2-transgenic mouse model. Collectively, these data show that HD-MAP delivery of a SARS-CoV-2 vaccine was superior to traditional needle-and-syringe vaccination and may be a significant addition to the ongoing COVID-19 (coronavirus disease 2019) pandemic.

Cellular responses at the application site of a high-density microarray patch delivering an influenza vaccine in a randomized, controlled phase I clinical trial.

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.

A chimeric dengue virus vaccine candidate delivered by high density microarray patches protects against infection in mice.

Dengue viruses (DENV) cause an estimated 390 million infections globally. With no dengue-specific therapeutic treatment currently available, vaccination is the most promising strategy for its control. A wide range of DENV vaccines are in development, with one having already been licensed, albeit with limited distribution. We investigated the immunogenicity and protective efficacy of a chimeric virus vaccine candidate based on the insect-specific flavivirus, Binjari virus (BinJV), displaying the structural prM/E proteins of DENV (BinJ/DENV2-prME). In this study, we immunized AG129 mice with BinJ/DENV2-prME via a needle-free, high-density microarray patch (HD-MAP) delivery system. Immunization with a single, 1 µg dose of BinJ/DENV2-prME delivered via the HD-MAPs resulted in enhanced kinetics of neutralizing antibody induction when compared to needle delivery and complete protection against mortality upon virus challenge in the AG129 DENV mouse model.

Safety, tolerability, and immunogenicity of influenza vaccination with a high-density microarray patch: Results from a randomized, controlled phase I clinical trial.

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.

Efficient Delivery of Dengue Virus Subunit Vaccines to the Skin by Microprojection Arrays.

Dengue virus is the most important arbovirus impacting global human health, with an estimated 390 million infections annually, and over half the world's population at risk of infection. While significant efforts have been made to develop effective vaccines to mitigate this threat, the task has proven extremely challenging, with new approaches continually being sought. The majority of protective, neutralizing antibodies induced during infection are targeted by the envelope (E) protein, making it an ideal candidate for a subunit vaccine approach. Using truncated, recombinant, secreted E proteins (sE) of all 4 dengue virus serotypes, we have assessed their immunogenicity and protective efficacy in mice, with or without Quil-A as an adjuvant, and delivered via micropatch array (MPA) to the skin in comparison with more traditional routes of immunization. The micropatch contains an ultra-high density array (21,000/cm2) of 110 µm microprojections. Mice received 3 doses of 1 µg (nanopatch, intradermal, subcutaneous, or intra muscular injection) or 10 µg (intradermal, subcutaneous, or intra muscular injection) of tetravalent sE spaced 4 weeks apart. When adjuvanted with Quil-A, tetravalent sE vaccination delivered via MPA resulted in earlier induction of virus-neutralizing IgG antibodies for all four serotypes when compared with all of the other vaccination routes. Using the infectious dengue virus AG129 mouse infectious dengue model, these neutralizing antibodies protected all mice from lethal dengue virus type 2 D220 challenge, with protected animals showing no signs of disease or circulating virus. If these results can be translated to humans, MPA-delivered sE represents a promising approach to dengue virus vaccination.

Microprojection arrays applied to skin generate mechanical stress, induce an inflammatory transcriptome and cell death, and improve vaccine-induced immune responses.

Chemical adjuvants are typically used to improve immune responses induced by immunisation with protein antigens. Here we demonstrate an approach to enhance immune responses that does not require chemical adjuvants. We applied microprojection arrays to the skin, producing a range of controlled mechanical energy to invoke localised inflammation, while administering influenza split virus protein antigen. We used validated computational modelling methods to identify links between mechanical stress and energy generated within the skin strata and resultant cell death. We compared induced immune responses to those induced by needle-based intradermal antigen delivery and used a systems biology approach to examine the nature of the induced inflammatory response, and correlated this with markers of cell stress and death. Increasing the microprojection array application energy and the addition of QS-21 adjuvant were each associated with enhanced antibody response to delivered antigen and with induction of gene transcriptions associated with TNF and NF-κB signalling pathways. We concluded that microprojection intradermal antigen delivery inducing controlled local cell death could potentially replace chemical adjuvants to enhance the immune response to protein antigen.

Safety, tolerability, acceptability and immunogenicity of an influenza vaccine delivered to human skin by a novel high-density microprojection array patch (Nanopatch™).

BACKGROUND: Injection using needle and syringe (N&S) is the most widely used method for vaccination, but requires trained healthcare workers. Fear of needles, risk of needle-stick injury, and the need to reconstitute lyophilised vaccines, are also drawbacks. The Nanopatch (NP) is a microarray skin patch comprised of a high-density array of microprojections dry-coated with vaccine that is being developed to address these shortcomings. Here we report a randomised, partly-blinded, placebo-controlled trial that represents the first use in humans of the NP to deliver a vaccine. METHODS: Healthy volunteers were vaccinated once with one of the following: (1) NPs coated with split inactivated influenza virus (A/California/07/2009 [H1N1], 15 µg haemagglutinin (HA) per dose), applied to the volar forearm (NP-HA/FA), n = 15; (2) NPs coated with split inactivated influenza virus (A/California/07/2009 [H1N1], 15 µg HA per dose), applied to the upper arm (NP-HA/UA), n = 15; (3) Fluvax® 2016 containing 15 µg of the same H1N1 HA antigen injected intramuscularly (IM) into the deltoid (IM-HA/D), n = 15; (4) NPs coated with excipients only, applied to the volar forearm (NP-placebo/FA), n = 5; (5) NPs coated with excipients only applied to the upper arm (NP-placebo/UA), n = 5; or (6) Saline injected IM into the deltoid (IM-placebo/D), n = 5. Antibody responses at days 0, 7, and 21 were measured by haemagglutination inhibition (HAI) and microneutralisation (MN) assays. FINDINGS: NP vaccination was safe and acceptable; all adverse events were mild or moderate. Most subjects (55%) receiving patch vaccinations (HA or placebo) preferred the NP compared with their past experience of IM injection with N&S (preferred by 24%). The antigen-vaccinated groups had statistically higher HAI titres at day 7 and 21 compared with baseline (p < 0.0001), with no statistical differences between the treatment groups (p > 0.05), although the group sizes were small. The geometric mean HAI titres at day 21 for the NP-HA/FA, NP-HA/UA and IM-HA/D groups were: 335 (189-593 95% CI), 160 (74-345 95% CI), and 221 (129-380 95% CI) respectively. A similar pattern of responses was seen with the MN assays. Application site reactions were mild or moderate, and more marked with the influenza vaccine NPs than with the placebo or IM injection. INTERPRETATION: Influenza vaccination using the NP appeared to be safe, and acceptable in this first time in humans study, and induced similar immune responses to vaccination by IM injection.

High-density microprojection array delivery to rat skin of low doses of trivalent inactivated poliovirus vaccine elicits potent neutralising antibody responses.

To secure a polio-free world, the live attenuated oral poliovirus vaccine (OPV) will eventually need to be replaced with inactivated poliovirus vaccines (IPV). However, current IPV delivery is less suitable for campaign use than OPV, and more expensive. We are progressing a microarray patch delivery platform, the Nanopatch, as an easy-to-use device to administer vaccines, including IPV. The Nanopatch contains an ultra-high density array (10,000/cm2) of short (~230 µm) microprojections that delivers dry coated vaccine into the skin. Here, we compare the relative immunogenicity of Nanopatch immunisation versus intramuscular injection in rats, using monovalent and trivalent formulations of IPV. Nanopatch delivery elicits faster antibody response kinetics, with high titres of neutralising antibody after just one (IPV2) or two (IPV1 and IPV3) immunisations, while IM injection requires two (IPV2) or three (IPV1 and IPV3) immunisations to induce similar responses. Seroconversion to each poliovirus type was seen in 100% of rats that received ~1/40th of a human dose of IPV delivered by Nanopatch, but not in rats given ~1/8th or ~1/40th dose by IM injection. Ease of administration coupled with dose reduction observed in this study suggests the Nanopatch could facilitate inexpensive IPV vaccination in campaign settings.

Potent response of QS-21 as a vaccine adjuvant in the skin when delivered with the Nanopatch, resulted in adjuvant dose sparing.

Adjuvants play a key role in boosting immunogenicity of vaccines, particularly for subunit protein vaccines. In this study we investigated the induction of antibody response against trivalent influenza subunit protein antigen and a saponin adjuvant, QS-21. Clinical trials of QS-21 have demonstrated the safety but, also a need of high dose for optimal immunity, which could possibly reduce patient acceptability. Here, we proposed the use of a skin delivery technology - the Nanopatch - to reduce both adjuvant and antigen dose but also retain its immune stimulating effects when compared to the conventional needle and syringe intramuscular (IM) delivery. We have demonstrated that Nanopatch delivery to skin requires only 1/100(th) of the IM antigen dose to induce equivalent humoral response. QS-21 enhanced humoral response in both skin and muscle route. Additionally, Nanopatch has demonstrated 30-fold adjuvant QS-21 dose sparing while retaining immune stimulating effects compared to IM. QS-21 induced localised, controlled cell death in the skin, suggesting that the danger signals released from dead cells contributed to the enhanced immunogenicity. Taken together, these findings demonstrated the suitability of reduced dose of QS-21 and the antigen using the Nanopatch to enhance humoral responses, and the potential to increase patient acceptability of QS-21 adjuvant.

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.

The changing shape of vaccination: improving immune responses through geometrical variations of a microdevice for immunization.

Micro-device use for vaccination has grown in the past decade, with the promise of ease-of-use, painless application, stable solid formulations and greater immune response generation. However, the designs of the highly immunogenic devices (e.g. the gene gun, Nanopatch or laser adjuvantation) require significant energy to enter the skin (30-90 mJ). Within this study, we explore a way to more effectively use energy for skin penetration and vaccination. These modifications change the Nanopatch projections from cylindrical/conical shapes with a density of 20,000 per cm(2) to flat-shaped protrusions at 8,000 per cm(2), whilst maintaining the surface area and volume that is placed within the skin. We show that this design results in more efficient surface crack initiations, allowing the energy to be more efficiently be deployed through the projections into the skin, with a significant overall increase in penetration depth (50%). Furthermore, we measured a significant increase in localized skin cell death (>2 fold), and resultant infiltrate of cells (monocytes and neutrophils). Using a commercial seasonal trivalent human influenza vaccine (Fluvax 2014), our new patch design resulted in an immune response equivalent to intramuscular injection with approximately 1000 fold less dose, while also being a practical device conceptually suited to widespread vaccination.

Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns.

Formulations for microprojection/microneedle vaccine delivery: Structure, strength and release profiles.

To develop novel methods for vaccine delivery, the skin is viewed as a high potential target, due to the abundance of immune cells that reside therein. One method, the use of dissolving microneedle technologies, has the potential to achieve this, with a range of formulations now being employed. Within this paper we assemble a range of methods (including FT-FIR using synchrotron radiation, nanoindentation and skin delivery assays) to systematically examine the effect of key bulking agents/excipients - sugars/polyols - on the material form, structure, strength, failure properties, diffusion and dissolution for dissolving microdevices. We investigated concentrations of mannitol, sucrose, trehalose and sorbitol from 1:1 to 30:1 with carboxymethylcellulose (CMC), although mannitol did not form our micro-structures so was discounted early in the study. The other formulations showed a variety of crystalline (sorbitol) and amorphous (sucrose, trehalose) structures, when investigated using Fourier transform far infra-red (FT-FIR) with synchrotron radiation. The crystalline structures had a higher elastic modulus than the amorphous formulations (8-12GPa compared to 0.05-11GPa), with sorbitol formulations showing a bimodal distribution of results including both amorphous and crystalline behaviour. In skin, diffusion properties were similar among all formulations with dissolution occurring within 5s for our small projection array structures (~100µm in length). Overall, slight variations in formulation can significantly change the ability of our projections to perform their required function, making the choice of bulking/vaccine stabilising agents of great importance for these devices.

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.

Depth-resolved characterization of diffusion properties within and across minimally-perturbed skin layers.

We examine by both experimental and computational means the diffusion of macromolecules through the skin strata (both the epidermis and dermis). Using mouse skin as a test case, we present a novel high-resolution technique to characterize the diffusion properties of heterogeneous biomaterials using 3D imaging of fluorescent probes, precisely-deposited in minimally-perturbed in vivo skin layers. We find the diffusivity of the delivered macromolecules (70 kDa and 2 MDa rhodamine-dextrans) low within the packed cellular arrangement of the epidermis, while gradually increasing (by ~an order of magnitude) through the dermis--as pores in the fibrillar network enlarge from the papillary to the reticular dermis. Our experimental and computational approaches for investigating the diffusion through skin strata help in the assessment and optimization of controlled delivery of drugs (e.g. vaccines) to specific sites (e.g. antigen presenting cells).

Induction of potent CD8⁺ T cell responses through the delivery of subunit protein vaccines to skin antigen-presenting cells using densely packed microprojection arrays.

The generation of both antibody and CD8⁺ T cell responses against pathogens is considered important for many advanced vaccines for diseases including tuberculosis, HIV and malaria. However, most current vaccines are delivered into muscle by the needle and syringe method and induce protection via humoral (antibody) immune responses. In this paper, we test the hypothesis that delivering a model subunit protein antigen (ovalbumin) to the skin's abundant immune cell population using a densely packed microprojection array (Nanopatch) enhances CD8⁺ T cell responses. We found that the Nanopatch significantly enhanced the CD8⁺ T cell responses when compared to intramuscular delivery of both antigen-only and adjuvanted cases (Quil-A and CpG; separately). To our knowledge, this is the first published study demonstrating significantly improved CD8⁺ T cell responses achieved by delivering subunit vaccines to the skin's abundant immune cell population. Successfully replicating these findings in humans could significantly advance the reach of vaccines.