Dynamics and turnover of memory CD8 T cell responses following yellow fever vaccination.

Understanding how immunological memory lasts a lifetime requires quantifying changes in the number of memory cells as well as how their division and death rates change over time. We address these questions by using a statistically powerful mixed-effects differential equations framework to analyze data from two human studies that follow CD8 T cell responses to the yellow fever vaccine (YFV-17D). Models were first fit to the frequency of YFV-specific memory CD8 T cells and deuterium enrichment in those cells 42 days to 1 year post-vaccination. A different dataset, on the loss of YFV-specific CD8 T cells over three decades, was used to assess out of sample predictions of our models. The commonly used exponential and bi-exponential decline models performed relatively poorly. Models with the cell loss following a power law (exactly or approximately) were most predictive. Notably, using only the first year of data, these models accurately predicted T cell frequencies up to 30 years post-vaccination. Our analyses suggest that division rates of these cells drop and plateau at a low level (0.1% per day, ∼ double the estimated values for naive T cells) within one year following vaccination, whereas death rates continue to decline for much longer. Our results show that power laws can be predictive for T cell memory, a finding that may be useful for vaccine evaluation and epidemiological modeling. Moreover, since power laws asymptotically decline more slowly than any exponential decline, our results help explain the longevity of immune memory phenomenologically.

Immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge.

Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 microg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.

Serological memory and long-term protection to novel H1N1 influenza virus after skin 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.

Ultrasound-enhanced chemotherapy and gene delivery for glioma cells.

Treatment of brain cancer is limited in part by inefficient intracellular delivery of drugs and DNA for chemotherapy and gene therapy, respectively. This study tested the hypothesis that ultrasound may be used to enhance intracellular delivery and efficacy of chemotherapeutics and genes in glioma cells in vitro. First, suitable ultrasound conditions were identified by measuring intracellular uptake of calcein and viability of GS 9L rat gliosarcoma cells after a range of different ultrasound exposures. We selected sonication at 10 J/cm2, which achieved intracellular delivery of approximately 10(6) molecules/cell. Next, glial cells were sonicated with varying concentrations of model chemotherapeutics: BCNU and bleomycin. For both drugs, cytotoxicity was increased in a synergistic manner when accompanied by ultrasound exposure. Finally, expression of a plasmid DNA encoding a GFP reporter was increased up to 30-fold when exposed to ultrasound. Altogether, these findings suggest that ultrasound may be useful to increase the efficacy of chemotherapy and gene therapy of glioma cells.

Physical parameters influencing optimization of ultrasound-mediated DNA transfection.

Ultrasound (US) has been shown to transiently disrupt cell membranes and, thereby, facilitate the loading of drugs and genes into viable cells. To address optimization of gene therapy applications, the aim of this work was to systematically determine the influence of physical parameters on transfection and viability of DU145 prostate cancer cells by two different DNA plasmids (pEGFP-N1 and pGL3). By sonicating cells in vitro in the presence of naked DNA, we found that transfection efficiency was increased by: 1. optimizing acoustic energy at 10 to 30 J/cm(2) (for our apparatus, at pressures above the cavitation threshold); 2. using 500-kHz US in the presence of Optison to nucleate cavition, rather than 24-kHz US without Optison; 3. increasing cell concentration from 10(6) to 10(7) cells/mL; and 4. changing temperature during sonication from 21 to 37 degrees C. The best conditions in this study increased transfection by almost 100-fold in the absence of significant DNA damage. Additional measurements indicated that less than one fourth of cells with DNA plasmid uptake into the cytosol showed DNA expression, which suggests that further optimizing transfection by US may require facilitating intracellular DNA trafficking.

Delivery of salmon calcitonin using a microneedle patch.

Peptides and polypeptides have important pharmacological properties but only a limited number have been exploited as therapeutics because of problems related to their delivery. Most of these drugs require a parenteral delivery system which introduces the problems of pain, possible infection, and expertise required to carry out an injection. The aim of this study was to develop a transdermal patch containing microneedles (MNs) coated with a peptide drug, salmon calcitonin (sCT), as an alternative to traditional subcutaneous and nasal delivery routes. Quantitative analysis of sCT after coating and drying onto microneedles was performed with a validated HPLC method. In vivo studies were carried out on hairless rats and serum levels of sCT were determined by ELISA. The AUC value of MNs coated with a trehalose-containing formulation (250 ± 83 ng/mL min) was not significantly different as compared to subcutaneous injections (403 ± 253 ng/mL min), but approximately 13 times higher than nasal administration (18.4 ± 14.5 ng/mL min). T(max) (7.5 ± 5 min) values for MN mediated administration were 50% shorter than subcutaneous injections (15 min), possibly due to rapid sCT dissolution and absorption by dermal capillaries. These results suggest that with further optimization of coating formulations, microneedles may enable administration of sCT and other peptides without the need for hypodermic injections.

Local response to microneedle-based influenza immunization in the skin.

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.

Effect of adjuvants on responses to skin immunization by microneedles coated with influenza subunit vaccine.

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.

Delivery of subunit influenza vaccine to skin with microneedles improves immunogenicity and long-lived protection.

Influenza infection represents a major socio-economic burden worldwide. Novel delivery methods can render influenza vaccination easier and more acceptable by the public, and importantly confer protection equal or superior to that induced by conventional systemic administration. An attractive target for vaccine delivery is the skin. Recent studies have demonstrated improved immune responses after transdermal delivery of inactivated influenza virus with microneedle patches. Here we show that immunization with a licensed influenza subunit vaccine coated on metal microneedles can activate both humoral and cellular arms of the immune response and confer improved long-term protection in the mouse model when compared to the conventional systemic route of delivery. These results demonstrate the promising potential of microneedle delivery of licensed influenza subunit vaccines, that could be beneficial in increasing vaccine coverage and protection and reducing influenza-related mortality worldwide.

Transdermal influenza immunization with vaccine-coated microneedle arrays.

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.

Electrosonic ejector microarray for drug and gene delivery.

We report on development and experimental characterization of a novel cell manipulation device-the electrosonic ejector microarray-which establishes a pathway for drug and/or gene delivery with control of biophysical action on the length scale of an individual cell. The device comprises a piezoelectric transducer for ultrasound wave generation, a reservoir for storing the sample mixture and a set of acoustic horn structures that form a nozzle array for focused application of mechanical energy. The nozzles are micromachined in silicon or plastic using simple and economical batch fabrication processes. When the device is driven at a particular resonant frequency of the acoustic horn structures, the sample mixture of cells and desired transfection agents/molecules suspended in culture medium is ejected from orifices located at the nozzle tips. During sample ejection, focused mechanical forces (pressure and shear) are generated on a microsecond time scale (dictated by nozzle size/geometry and ejection velocity) resulting in identical "active" microenvironments for each ejected cell. This process enables a number of cellular bioeffects, from uptake of small molecules and gene delivery/transfection to cell lysis. Specifically, we demonstrate successful calcein uptake and transfection of DNA plasmid encoding green fluorescent protein (GFP) into human malignant glioma cells (cell line LN443) using electrosonic microarrays with 36, 45 and 50 mum diameter nozzle orifices and operating at ultrasound frequencies between 0.91 and 0.98 MHz. Our results suggest that efficacy and the extent of bioeffects are mainly controlled by nozzle orifice size and the localized intensity of the applied acoustic field.

Mechanosensing using drag force for imaging soft biological membranes.

We investigate physical processes taking place during nanoscale mechanosensing of soft biological membranes in liquid environments. Examples include tapping mode imaging by atomic force microscope (AFM) and microscopy based on the Brownian motion of a nanoparticle in an optical-tweezers-controlled trap. The softness and fluidity of the cellular membrane make it difficult to accurately detect (i.e., image) the shape of the cell using traditional mechanosensing methods. The aim of the reported work is to theoretically evaluate whether the drag force acting on the nanoscale mechanical probe due to a combined effect of intra- and extracellular environments can be exploited to develop a new imaging mode suitable for soft cellular interfaces. We approach this problem by rigorous modeling of the fluid mechanics of a complex viscoelastic biosystem in which the probe sensing process is intimately coupled to the membrane biomechanics. The effects of the probe dimensions and elastic properties of the membrane as well as intra- and extracellular viscosities are investigated in detail to establish the structure and evolution of the fluid field as well as the dynamics of membrane deformation. The results of numerical simulations, supported by predictions of the scaling analysis of forces acting on the probe, suggest that viscous drag is the dominant force dictating the probe dynamics as it approaches a biological interface. The increase in the drag force is shown to be measurable, to scale linearly with an increase in the viscosity ratio of the fluids on either side of the membrane, and to be inversely proportional to the probe-to-membrane distance. This leads to the postulation of a new strategy for lipid membrane imaging by AFM or other mechanosensing methods using a variation in the maximum drag force as an indicator of the membrane position.