Frances E. Pearson

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

Background Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8+ T cell responses to a malaria antigen induced by a live vaccine. Methodology and Findings Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. Conclusions/Significance This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8+ T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes.

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 (i.m.) or intradermal (i.d.) injection. However, the mechanisms behind 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-IgG endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, co-localization 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.

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 cm2 to flat-shaped protrusions at 8,000 per cm2, 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.

Functional anti-polysaccharide IgG titres induced by unadjuvanted pneumococcal-conjugate vaccine when delivered by microprojection-based skin patch

Adequate access to effective and affordable vaccines is essential for the prevention of mortality due to infectious disease. Pneumonia--a consequence of Streptococcus pneumoniae infection--is the worlds leading cause of death in children aged under 5 years. The development of a needle-free, thermostable pneumococcal-conjugate vaccine (PCV) could revolutionise the field by reducing cold-chain and delivery constraints. Skin patches have been used to deliver a range of vaccines, with some inducing significantly higher vaccine-specific immunogenicity than needle-injected controls in pre-clinical models, though they have yet to be used to deliver a PCV. We dry-coated a licensed PCV onto a microprojection-based patch (the Nanopatch) and delivered it to mouse skin. We analysed resulting anti-polysaccharide IgG responses. With and without adjuvant, anti-polysaccharide IgG titres induced by Nanopatch immunisation were significantly higher than dose-matched intramuscular controls. These improved responses were primarily obtained against pneumococcal serotypes 4 and 14. Importantly, capsule-specific IgG correlated with functionality in an opsonophagocytic killing assay. We demonstrate enhanced anti-PCV immunogenicity when delivered by Nanopatch over intramuscular injection. As the first study of a PCV delivered by a skin vaccination technology, this report indicates the potential for reduced costs and greater global distribution of such a vaccine.

Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells

Dendritic cells (DC) initiate the differentiation of CD4+ helper T cells into effector cells including Th1 and Th17 responses that play an important role in inflammation and autoimmune disease pathogenesis. In mice, Th1 and Th17 responses are regulated by different conventional (c) DC subsets, with cDC1 being the main producers of IL-12p70 and inducers of Th1 responses, while cDC2 produce IL-23 to promote Th17 responses. The role that human DC subsets play in memory CD4+ T cell activation is not known. This study investigated production of Th1 promoting cytokine IL-12p70, and Th17 promoting cytokines, IL-1β, IL-6, and IL-23, by human blood monocytes, CD1c+ DC, CD141+ DC, and plasmacytoid DC and examined their ability to induce Th1 and Th17 responses in memory CD4+ T cells. Human CD1c+ DC produced IL-12p70, IL-1β, IL-6, and IL-23 in response to R848 combined with LPS or poly I:C. CD141+ DC were also capable of producing IL-12p70 and IL-23 but were not as proficient as CD1c+ DC. Activated CD1c+ DC were endowed with the capacity to promote both Th1 and Th17 effector function in memory CD4+ T cells, characterized by high production of interferon-γ, IL-17A, IL-17F, IL-21, and IL-22. These findings support a role for CD1c+ DC in autoimmune inflammation where Th1/Th17 responses play an important role in disease pathogenesis.

Activation of human CD141+ and CD1c+ dendritic cells in vivo with combined TLR3 and TLR7/8 ligation

Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141+ and CD1c+ myeloid and CD123+ plasmacytoid dendritic cells (DC) develop from human cord blood CD34+ cells in immunodeficient mice. CD141+ DC are the human equivalents of murine CD8+/CD103+ DC which are essential for the induction of tumor‐inhibitory cytotoxic T lymphocyte responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34+‐engrafted NSG‐A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogs polyinosinic‐polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8, respectively, both of which are expressed by CD141+ DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c+ DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross‐presentation and the induction of cytotoxic T lymphocyte responses including IFN‐α, IFN‐β, IL‐12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy.