Celia L. McNeilly

Improved DNA vaccination by skin-targeted delivery using dry-coated densely-packed microprojection arrays

HSV-2-gD2 DNA vaccine was precisely delivered to immunologically sensitive regions of the skin epithelia using dry-coated microprojection arrays. These arrays delivered a vaccine payload to the epidermis and the upper dermis of mouse skin. Immunomicroscopy results showed that, in 43 ± 5% of microprojection delivery sites, the DNA vaccine was delivered to contact with professional antigen presenting cells in the epidermal layer. Associated with this efficient delivery of the vaccine into the vicinity of the professional antigen presenting cells, we achieved superior antibody responses and statistically equal protection rate against an HSV-2 virus challenge, when compared with the mice immunized with intramuscular injection using needle and syringe, but with less than 1/10th of the delivered antigen.

Towards the Immunoproteome of Neisseria meningitidis

Despite the introduction of conjugated polysaccharide vaccines for many of the Neisseria meningitidis serogroups, neisserial infections continue to cause septicaemia and meningitis across the world. This is in part due to the difficulties in developing a, cross-protective vaccine that is effective against all serogroups, including serogroup B meningococci. Although convalescent N. meningitidis patients develop a natural long-lasting cross-protective immunity, the antigens that mediate this response remain unknown. To help define the target of this protective immunity we identified the proteins recognized by IgG in sera from meningococcal patients by a combination of 2D protein gels, western blots and mass spectrometry. Although a number of outer membrane antigens were identified the majority of the antigens were cytoplasmic, with roles in cellular processes and metabolism. When recombinant proteins were expressed and used to raise sera in mice, none of the antigens elicited a positive SBA result, however flow cytometry did demonstrate that some, including the ribosomal protein, RplY were localised to the neisserial cell surface.

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.

Microprojection arrays to immunise at mucosal surfaces.

The buccal mucosa (inner cheek) is an attractive site for delivery of immunotherapeutics, due to its ease of access and rich antigen presenting cell (APC) distribution. However, to date, most delivery methods to the buccal mucosa have only been topical-with the challenges of: 1) an environment where significant biomolecule degradation may occur; 2) inability to reach the APCs that are located deep in the epithelium and lamina propria; and 3) salivary flow and mucous secretion that may result in removal of the therapeutic agent before absorption has taken place. To overcome these challenges and achieve consistent, repeatable targeted delivery of immunotherapeutics to within the buccal mucosa (not merely on to the surface), we utilised microprojection arrays (Nanopatches-110 μm length projections, 3364 projections, 16 mm2 surface area) with a purpose built clip applicator. The mechanical application of Nanopatches bearing a dry-coated vaccine (commercial influenza vaccine, as a test case immunotherapeutic) released the vaccine to a depth of 47.8±14.8 μm (mean±SD, n=4), in the mouse buccal mucosa (measured using fluorescent delivered dyes and CryoSEM). This location is in the direct vicinity of APCs, facilitating antigenic uptake. Resultant systemic immune responses were similar to systemic immunization methods, and superior to comparative orally immunised mice. This confirms the Nanopatch administered vaccine was delivered into the buccal mucosa and not ingested. This study demonstrates a minimally-invasive delivery device with rapid (2 min of application time), accurate and consistent release of immunotherapeutics in to the buccal mucosa-that conceptually can be extended in to human use for broad and practical utility.

Collagen deposition assessment in burn scar tissue using second harmonic generation and multi-photon microscopy

S2 Journal of Investigative Dermatology (2010), Volume 130 007 Hypothyroidism improves random-pattern skin fl ap survival in rats Sina Rahimpour1, Behtash G. Nezami1, Negin Karimian1, Maryam SotoudehAnvari2, Sara Khalaj1, Laleh Montaser-Kouhsari1, Ahmadreza Dehpour1 1Pharmacology department, school of medicine,Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic of, 2Department of surgical and clinical pathology,Terhan Heart Center,Terhan University of Medical Sciences,Tehran, Iran, Islamic Republic of The protective effect of hypothyroidism against ischemic or toxic conditions is shown in various tissues. We investigated the effect of hypothyroidism and acute local effect of propylthiouracil (PTU) and methimazole (MMI) on the outcome of lethal ischemia in this fl ap model. Forty-two Sprauge-Dawley rats were randomly divided into 7 groups. In all groups, dorsal fl aps with caudal pedicles were elevated at midline and fl ap survival was measured at the seventh day after surgery. The fi rst group served as control and received 1 ml of 0.9% saline solution into their fl ap before fl ap elevation. In groups 2 and 3, hypothyroidism was induced by administration of either PTU 0.05% or MMI 0.04% in their drinking water for four weeks. Next four groups received local injections of MMI (10, 20, 50 or 100 μg/fl ap) before fl ap elevation. Local PTU injection was ignored due to insolubility of the agent. Hypothyroidism was induced in chronic PTU and MMI treated groups, and animals in these groups showed signifi cant increase in their fl ap survival compared to control euthyroid rats (79.47 ± 10.49% and 75.48 ± 12.93% vs. 52.26 ± 5.75%, respectively, P< 0.01). Acute local treatment of skin fl aps with MMI failed to signifi cantly affect the fl ap survival. This study demonstrates for the fi rst time that hypothyroidism survives random-pattern skin fl aps in rats. 008 VEGF plays a key role enhancing epidermal and blood vessel protection against stress Ludivine Mur1, Cedric Pouzet1, Catherine Serre1, Catherine Gondran1, Eric Bauza1, Jean Marie Botto1, Claude Dal Farra2, Nouha Domloge1 1Vincience ISP Global Skin Research Center, Sophia Antipolis, France, 2ISP Corporate Research Center, Wayne, United States Vascular endothelial growth factor (VEGF) is a crucial element of endothelial cells and angiogenesis and plays diverse roles in skin photoaging, hypoxia, and wound healing. We investigated the expression of VEGF-A and VEGFR-2 (Flk-1), its major receptor, in different cell lines by RT-PCR. VEGF-A and Flk-1 immunofl uorescence studies showed that IV09.006, a compound designed to target VEGF pathway, increased the expression of these two proteins in normal human keratinocytes (NHK) and endothelial cells. Ex vivo studies showed that VEGF-A expression in the epidermis is mainly located in the suprabasal layers. UVB irradiation and H2O2 stresses increased VEGF-A expression in the epidermis. In parallel, pre-treatment with IV09.006 was shown to protect skin from stress damage. For in vivo evaluation, we used chicken chorio-allantoïc membrane (CAM). IV09.006 active was applied directly on the CAM for 24h, and then a stress induced by UVB irradiation (60 mJ/cm2) or H2O2 (10 mM) was applied. A time course observation of the blood vessel network was performed after each stress condition. Our study showed that pre-induction of VEGF-A and Flk-1 enabled a better maintenance of the blood vessel network, preventing vasodilatation and coagulation induced by stress. Taken together, these results indicate that the positive modulation of VEGF-A and Flk-1 expression could be linked to a better preservation of the epidermis from UVB and oxidative stress-induced damage, as well as a protection of the blood vessel network from these stresses. 009 [Oral 109] A unique population of infl ammatory macrophages induced by iron impairs cutaneous wound healing Anca Sindrilaru1, Thorsten Peters1, Corina Baican2, Johannes Weiss1, Meinhard Wlaschek1, Cord Sunderkötter3, Karin Scharffetter-Kochanek1 1University of Ulm, Dept of Dermatology & Allergic Diseases, Germany, 2University of Cluj-Napoca, Dept of Dermatology & Venerology, Cluj-Napoca, Romania, 3University of Münster, Dept of Dermatology, Germany Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic infl ammatory disorders like arteriosclerosis, multiple sclerosis and chronic venous leg ulcers (CVU). However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic infl ammation and affect resident fi broblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with CVU, a model disease for macrophage-driven chronic infl ammation, while establishing a murine model closely refl ecting its pathogenesis. Here we show that iron overloading of macrophages in CVU and in irondextran-treated murine full-thickness excisional wounds induce a novel macrophage population in situ which up-regulate CD163, the hemoglobin-haptoglobin scavenger receptor for iron. CD163hi wound macrophages mount a persistent pro-infl ammatory activation state with high expression of M1 markers (TNFahighIL-12highCD11bhighCCR2high) and the concomitant intermediate expression of anti-infl ammatory M2 markers (IL-4RamedDectin-1medCD36medCD206med), suggestive for an incomplete switch towards the tissue repair-promoting M2 activation phenotype. We show that ‘hybrid’ M1/M2 activated macrophages via enhanced TNFa and hydroxyl radicals release – perpetuate infl ammation and install a p16INK4a-dependent senescence program in resident fi broblasts eventually leading to tissue breakdown and impaired wound healing. Understanding the role of macrophage activation for persistency or resolution of infl ammation in chronic venous ulcers and other chronic infl ammatory diseases holds substantial promise for the development of novel therapies for these diffi cult-to-treat conditions. 010 [Oral 110] Toll-like receptor signaling in dendritic cells is suffi cient to mediate Imiquimodinduced psoriasis-like skin infl ammation in mice Christian Wohn1, Errol Prens2, Sabina Onderwater1, Edwin Florencia2, Heike Weighardt3, Björn Clausen1 1Dept of Immunology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 2Dept of Dermatology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 3Institut für Umweltmedizinische Forschung, Heinrich-Heine-Universität Düsseldorf, Germany Psoriasis is a common infl ammatory skin disease characterized by sharply demarcated chronic red plaques covered by white scales. Based on the observation that Imiquimod (IMQ) treatment leads to de novo development or relapse of psoriasis in patients, we established a new mouse model for human psoriasis. Daily application of IMQ onto mouse back skin induces infl amed scaly lesions resembling plaque type psoriasis. These lesions show increased epidermal proliferation, abnormal cell differentiation, neoangiogenesis and infi ltrates consisting of neutrophils, CD4+ T cells, conventional and plasmacytoid dendritic cells (DC). We previously demonstrated that lesion development is critically dependent on IL-23 and IL-17. However, the cell types triggering the infl ammatory process remain elusive. IMQ activates diverse cells of the immune system after binding to toll-like receptors (TLR)-7/8. To investigate the role of different (skin) DC in initiating IMQ-induced psoriasis, we generated MyD88-defi cient mice in which TLR-signaling can be conditionally switched on by Cre-mediated excision of a stop cassette (MyD88stp/stp mice). As expected, MyD88stp/stp animals are resistant to IMQ-induced psoriasis. In contrast, mice with selective reconstitution of TLR signaling in all CD11c+ DC acquire full-blown psoriasiform skin disease following IMQ painting. Intriguingly, mice with TLR signaling restricted to Langerin+ DC subsets, including epidermal Langerhans cells and Langerin+ dermal DC, develop attenuated disease. These data imply that DC are suffi cient to mediate IMQ-induced psoriasis. In ongoing experiments we are further dissecting the requirement of different DC subsets for the induction of disease. 011 [Oral 063] Human IL-10 producing regulatory B cells control CD4+ T cell proliferation Jean-David Bouaziz1, Sébastien Calbo2,3, Maud Maho-Vaillant2, Anne Saussine1, Martine Bagot1, Armand Bensussan1, Philippe Musette2 1INSERM U976, Saint Louis Hospital, Paris, France, 2INSERM U905, Charles Nicolle Hospital, Rouen, France, 3Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore The existence of interleukin 10 (IL-10) producing regulatory B cells that downmodulate infl ammation has already been validated in mice. Especially, a potent subset of regulatory B cells with a CD1dhighCD5+CD19high phenotype was found to decrease contact hypersensitivity in an IL-10-dependent manner. In humans, a transitional B cell subset has recently been shown to exhibit regulatory capacities in vitro.We investigated the existence of IL-10 producing B cells in human blood and spleen and evaluated their phenotype. We also tested the optimal in vitro conditions to trigger IL-10 production by B cells and tested their capacities to regulate CD4+CD25T cell proliferation.We were able to detect after ex vivo short time stimulation, IL-10 producing B cells in human blood and spleen (1.8% and 1.1% of blood and spleen human B cells produced IL-10 as detected by intracellular cytokine staining). Blood IL-10 producing B cells were relatively enriched within the memory (CD27+) and transitional (CD38highCD24high) B cell subsets but IL-10 producing B cells were detected within the whole B cell lineage. Combined CpG-B and anti-immunoglobulin stimulation, rather than CD40L-CD40 pathway, was the most potent stimulus for inducing IL-10 secretion (ELISA assay). Under thes