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Unlabelled: Microneedle patches (MN) provide a novel method of vaccine delivery to the skin with the objective of targeting the large network of resident antigen-presenting cells to induce an efficient immune response. Our previous reports demonstrated that cutaneous delivery of inactivated influenza virus-coated MN to mice protects against lethal infection. Protection is correlated with sustained levels of anti-influenza virus serum antibodies, hemagglutination inhibition titers, and robust cellular responses that are often stronger than those generated by intramuscular vaccination. Here we dissect the early events occurring in murine skin after microneedle delivery of inactivated influenza virus. We demonstrate correlation of immunization against influenza virus with a local increase of cytokines important for recruitment of neutrophils, monocytes and dendritic cells at the site of immunization. We also observed prolonged antigen deposition, and migration of matured dendritic cells bearing influenza virus antigen from the skin., Importance: The immunological mechanisms by which MN vaccination confers protective immunity are not well understood. The present study provides a first analysis of the early immune events after microneedle-based vaccination.

Background: A major goal in influenza vaccine development is induction of serological memory and cellular responses to confer long-term protection and limit virus spread after infection. Here, we investigate induction of long-lived immunity against the 2009 H1N1 virus after skin vaccination., Methods: BALB/c mice received a single dose of 5 μg inactivated A/California/04/09 virus via coated metal microneedles (MN) applied to skin or via subcutaneous injection., Results: MN or subcutaneous vaccination elicited similar serum IgG and hemagglutination inhibition titers and 100% protection against lethal viral challenge 6 weeks after vaccination. Six months after vaccination, the subcutaneous group exhibited a 60% decrease in functional antibody titers and extensive lung inflammation after challenge with 10 × LD(50) of homologous virus. In contrast, the MN group maintained high functional antibody titers and IFN-γ levels, inhibition of viral replication, and no signs of lung inflammation after challenge. MN vaccination conferred complete protection against lethal challenge, whereas subcutaneous vaccination induced only partial protection. These findings were further supported by high numbers of bone marrow plasma cells and spleen antibody-secreting cells detected in the MN group., Conclusions: A single skin vaccination with MN induced potent long-lived immunity and improved protection against the 2009 H1N1 influenza virus, compared with subcutaneous injection.

Influenza prophylaxis would benefit from a vaccination method enabling simplified logistics and improved immunogenicity without the dangers posed by hypodermic needles. Here we introduce dissolving microneedle patches for influenza vaccination using a simple patch-based system that targets delivery to skin's antigen-presenting cells. Microneedles were fabricated using a biocompatible polymer encapsulating inactivated influenza virus vaccine for insertion and dissolution in the skin within minutes. Microneedle vaccination generated robust antibody and cellular immune responses in mice that provided complete protection against lethal challenge. Compared to conventional intramuscular injection, microneedle vaccination resulted in more efficient lung virus clearance and enhanced cellular recall responses after challenge. These results suggest that dissolving microneedle patches can provide a new technology for simpler and safer vaccination with improved immunogenicity that could facilitate increased vaccination coverage.

Background: Influenza is a contagious disease caused by a pathogenic virus, with outbreaks all over the world and thousands of hospitalizations and deaths every year. Due to virus antigenic drift and short-lived immune responses, annual vaccination is required. However, vaccine coverage is incomplete, and improvement in immunization is needed. The objective of this study is to investigate a novel method for transdermal delivery using metal microneedle arrays (MN) coated with inactivated influenza virus to determine whether this route is a simpler and safer approach than the conventional immunization, capable to induce robust immune responses and confer protection against lethal virus challenge., Methodology/principal Findings: Inactivated A/Aichi/2/68 (H3N2) influenza virus was coated on metal microneedle arrays and applied to mice as a vaccine in the caudal dorsal skin area. Substantial antibody titers with hemagglutination inhibition activity were detected in sera collected two and four weeks after a single vaccine dose. Challenge studies in mice with 5 x LD(50) of mouse adapted Aichi virus demonstrated complete protection. Microneedle vaccination induced a broad spectrum of immune responses including CD4+ and CD8+ responses in the spleen and draining lymph node, a high frequency of antigen-secreting cells in the lung and induction of virus-specific memory B-cells. In addition, the use of MN showed a dose-sparing effect and a strong Th2 bias when compared to an intramuscular (IM) reference immunization., Conclusions/significance: The present results show that delivery of inactivated influenza virus through the skin using metal microneedle arrays induced strong humoral and cellular immune responses capable of conferring protection against virus challenge as efficiently as intramuscular immunization, which is the standard vaccination route. In view of the convenience of delivery and the potential for self-administration, vaccine-coated metal microneedles may provide a novel and highly effective immunization method.

Objective: To extend our studies on the prolonged and differential effect of natalizumab on T lymphocyte numbers in the cerebrospinal fluid, we investigated the number and phenotypes of leukocytes and the expression of major histocompatibility complex (MHC) classes I and II in cerebral perivascular spaces (CPVS). We hypothesized that natalizumab reduces the number of antigen presenting cells in CPVS., Design: A case-control study in which inflammatory cell numbers in the CPVS of cerebral tissue were assessed by immunohistochemical staining., Subjects: A patient with multiple sclerosis (MS) who developed progressive multifocal leukoencephalopathy (PML) during natalizumab therapy. Controls included location-matched cerebral autopsy material of patients without disease of the central nervous system, patients with MS not treated with natalizumab, and patients with PML not associated with natalizumab therapy., Results: The absolute number of CPVS in the patient with MS treated with natalizumab was significantly lower than in the control groups owing to extensive destruction of the tissue architecture. The expression of MHC class II molecules and the number of CD209+ dendritic cells were significantly decreased in the CPVS of the patient with MS treated with natalizumab. No CD4+ T cells were detectable., Conclusions: Our observations may explain the differential and prolonged effects of natalizumab therapy on leukocyte numbers in the cerebrospinal fluid.

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

Protective protein/cathepsin A (PPCA) is a pleiotropic lysosomal enzyme that complexes with beta-galactosidase and neuraminidase, and possesses serine carboxypeptidase activity. Its deficiency in man results in the neurodegenerative lysosomal storage disorder galactosialidosis (GS). The mouse model of this disease resembles the human early onset phenotype and results in severe nephropathy and ataxia. To understand better the pathophysiology of the disease, we compared the occurrence of lysosomal PPCA mRNA and protein in normal adult mouse tissues with the incidence of lysosomal storage in PPCA(-/-) mice. PPCA expression was markedly variable among different tissues. Most sites that produced both mRNA and protein at high levels in normal mice showed extensive and overt storage in the knockout mice. However, this correlation was not consistent as some cells that normally expressed high levels of PPCA were unaffected in their storage capability in the PPCA(-/-) mice. In addition, some normally low expressing cells accumulated large amounts of undegraded products in the GS mouse. This apparent discrepancy may reflect a requirement for the catalytic rather than the protective function of PPCA and/or the presence of cell-specific substrates in certain cell types. A detailed map showing the cellular distribution of PPCA in nomal mouse tissues as well as the sites of lysosomal storage in deficient mice is critical for accurate assessment of the effects of therapeutic interventions.

Human GM1-gangliosidosis is caused by a genetic deficiency of lysosomal acid beta-galactosidase (beta-gal). The disease manifests itself either as an infantile, juvenile or adult form and is primarily a neurological disorder with progressive brain dysfunction. A mouse model lacking a functional beta-gal gene has been generated by homologous recombination and embryonic stem cell technology. Tissues from affected mice are devoid of beta-gal mRNA and totally deficient in GM1-ganglioside-hydrolyzing capacity. Storage material was already conspicuous in the brain at 3 weeks. By 5 weeks, extensive storage of periodic acid Schiff-positive material was observed in neurons throughout the brain and spinal cord. Consistent with the neuropathology, abnormal accumulation of GM1-ganglioside in the brain progressed from twice to almost five times the normal amount during the period from 3 weeks to 3.5 months. Despite the accumulation of brain GM1-ganglioside at the level equal to or exceeding that seen in gravely ill human patients, these mice show no overt clinical phenotype up to 4-5 months. However, tremor, ataxia and abnormal gait become apparent in older mice. Thus, the beta-gal-deficient mice appear to mimic closely the pathological, biochemical and clinical abnormalities of the human disease.