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

Abstract: 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

Microarray patch delivery of un-adjuvanted influenza vaccine induces potent and broad-spectrum immune responses in a phase I clinical trial

Microarray patches (MAPs) offer the possibility of improved vaccine thermostability and dose-sparing potential 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. Here, we report a phase I trial (ACTRN12618000112268/ U1111-1207-3550) using the Vaxxas high-density MAP (HD-MAP) to deliver a monovalent influenza vaccine to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs. To the best of our knowledge, this is the first study determining dose reduction potential using MAPs in humans. Monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/ 2015 [H1N1] haemagglutinin (HA) was delivered by MAP into the volar forearm or upper arm, or given intramuscularly (IM) once. Participants (20 per group) received HD-MAPs delivering doses of 15, 10, 5, 2.5 or 0 µg of HA or an IM injection of quadrivalent influenza vaccine (QIV). In two subgroups, skin biopsies were taken on days 1 (pre-vaccination) and 4 for analysis of the cellular composition from the HD-MAP application sites. All laboratory investigators were blind to treatment and participant allocation. The primary objectives of the study were safety and tolerability. Secondary objectives included immunogenicity and dose de-escalation assessments of the influenza vaccine delivered by HD-MAP. Both objectives were assessed for up to 60 days post-vaccination. Please click Download on the upper right corner to see the full abstract

Clinical and laboratory diagnosis of dengue virus infection

Infection with any of the 4 dengue virus serotypes results in a diverse range of symptoms, from mild undifferentiated fever to life-threatening hemorrhagic fever and shock. Given that dengue virus infection elicits such a broad range of clinical symptoms, early and accurate laboratory diagnosis is essential for appropriate patient management. Virus detection and serological conversion have been the main targets of diagnostic assessment for many years, however cross-reactivity of antibody responses among the flaviviruses has been a confounding issue in providing a differential diagnosis. Furthermore, there is no single, definitive diagnostic biomarker that is present across the entire period of patient presentation, particularly in those experiencing a secondary dengue infection. Nevertheless, the development and commercialization of point-of-care combination tests capable of detecting markers of infection present during different stages of infection (viral nonstructural protein 1 and immunoglobulin M) has greatly simplified laboratory-based dengue diagnosis. Despite these advances, significant challenges remain in the clinical management of dengue-infected patients, especially in the absence of reliable biomarkers that provide an effective prognostic indicator of severe disease progression. This review briefly summarizes some of the complexities and issues surrounding clinical dengue diagnosis and the laboratory diagnostic options currently available

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 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

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

Colocalization of cell death with antigen deposition in skin enhances vaccine immunogenicity

Vaccines delivered to the skin by microneedles - with and without adjuvants - have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular or intradermal injection. However, the mechanisms underlying this skin-mediated "adjuvant" effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (∼65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ∼1-10 MPa. Nanopatch-immunized groups also yielded consistently higher anti-immunoglobulin G endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, colocalization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a "physical immune enhancer" for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines

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/cm) 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