Design, Formulation, and Evaluation of Novel Dissolving Microarray Patches Containing Rilpivirine for Intravaginal Delivery.

Antiretroviral (ARV) drugs have, for many years, been studied and administered in the prevention and treatment of human immunodeficiency virus (HIV). Intramuscular (IM) injection of long acting (LA) ARVs are in clinical development, but injectable formulations require regular access to healthcare facilities and disposal facilities for sharps. The development of a discrete, self-administered, and self-disabling vehicle to deliver ARVs could obviate these issues. This study describes the formulation, mechanical characterization, and in vivo evaluation of dissolving microarray patches (MAPs) containing a LA nanosuspension of the ARV, rilpivirine (RPV, RPV LA), for vaginal delivery. This is the first study to apply MAPs into vaginal tissue. The RPV LA MAPs penetrate ex vivo skin and a synthetic vaginal skin model and withstand the effects of potential dragging motion across synthetic vaginal epithelium. In in vivo studies, the mean plasma concentration of RPV in rats at the 56 day endpoint (116.5 ng mL-1 ) is comparable to that achieved in the IM control cohort (118.9 ng mL-1 ). RPV is detected systemically, in lymph and vaginal tissue, indicating the potential to deliver RPV LA to primary sites of viral challenge and replication. This innovative research has future potential for patients and healthcare workers, particularly in low-resource settings.

Design, formulation and evaluation of novel dissolving microarray patches containing a long-acting rilpivirine nanosuspension.

One means of combating the spread of human immunodeficiency virus (HIV) is through the delivery of long-acting, antiretroviral (ARV) drugs for prevention and treatment. The development of a discreet, self-administered and self-disabling delivery vehicle to deliver such ARV drugs could obviate compliance issues with daily oral regimens. Alternatives in development, such as long-acting intramuscular (IM) injections, require regular access to health care facilities and disposal facilities for sharps. Consequently, this proof of concept study was developed to evaluate the use of dissolving microarray patches (MAPs) containing a long-acting (LA) nanosuspension of the candidate ARV drug, rilpivirine (RPV). MAPs were mechanically strong and penetrated skin in vitro, delivering RPV intradermally. In in vivo studies, the mean plasma concentration of RPV in rats (431 ng/ml at the Day 7 time point) was approximately ten-fold greater than the trough concentration observed after a single-dose in previous clinical studies. These results are the first to indicate, by the determination of relative exposures between IM and MAP administration, that larger multi-array dissolving MAPs could potentially be used to effectively deliver human doses of RPV LA. Importantly, RPV was also detected in the lymph nodes, indicating the potential to deliver this ARV agent into one of the primary sites of HIV replication over extended durations. These MAPs could potentially improve patient acceptability and adherence to HIV prevention and treatment regimens and combat instances of needle-stick injury and the transmission of blood-borne diseases, which would have far-reaching benefits, particularly to those in the developing world.

Cost of goods sold and total cost of delivery for oral and parenteral vaccine packaging formats.

Despite limitations of glass packaging for vaccines, the industry has been slow to implement alternative formats. Polymer containers may address many of these limitations, such as breakage and delamination. However, the ability of polymer containers to achieve cost of goods sold (COGS) and total cost of delivery (TCOD) competitive with that of glass containers is unclear, especially for cost-sensitive low- and lower-middle-income countries. COGS and TCOD models for oral and parenteral vaccine packaging formats were developed based on information from subject matter experts, published literature, and Kenya's comprehensive multiyear plan for immunization. Rotavirus and inactivated poliovirus vaccines (IPV) were used as representative examples of oral and parenteral vaccines, respectively. Packaging technologies evaluated included glass vials, blow-fill-seal (BFS) containers, preformed polymer containers, and compact prefilled auto-disable (CPAD) devices in both BFS and preformed formats. For oral vaccine packaging, BFS multi-monodose (MMD) ampoules were the least expensive format, with a COGS of $0.12 per dose. In comparison, oral single-dose glass vials had a COGS of $0.40. BFS MMD ampoules had the lowest TCOD of oral vaccine containers at $1.19 per dose delivered, and ten-dose glass vials had a TCOD of $1.61 per dose delivered. For parenteral vaccines, the lowest COGS was achieved with ten-dose glass vials at $0.22 per dose. In contrast, preformed CPAD devices had the highest COGS at $0.60 per dose. Ten-dose glass vials achieved the lowest TCOD of the parenteral vaccine formats at $1.56 per dose delivered. Of the polymer containers for parenteral vaccines, BFS MMD ampoules achieved the lowest TCOD at $1.89 per dose delivered, whereas preformed CPAD devices remained the most expensive format, at $2.25 per dose delivered. Given their potential to address the limitations of glass and reduce COGS and TCOD, polymer containers deserve further consideration as alternative approaches for vaccine packaging.

Transdermal delivery of vitamin K using dissolving microneedles for the prevention of vitamin K deficiency bleeding.

Vitamin K deficiency within neonates can result in vitamin K deficiency bleeding. Ensuring that newborns receive vitamin K is particularly critical in places where access to health care and blood products and transfusions is limited. The World Health Organization recommends that newborns receive a 1 mg intramuscular injection of vitamin K at birth. Evidence from multiple surveillance studies shows that the introduction of vitamin K prophylaxis reduces the incidence of vitamin K deficiency bleeding. Despite these recommendations, coverage of vitamin K prophylactic treatment in low-resource settings is limited. An intramuscular injection is the most common method of vitamin K administration in neonates. In low- and middle-income countries, needle sharing may occur, which may result in the spread of bloodborne diseases. The objective of our study was to investigate the manufacture of microneedles for the delivery of vitamin K. Following microneedle fabrication, we performed insertion studies to assess the microneedle's mechanical properties. Results indicate that vitamin K in a microneedle array was successfully delivered in vitro across neonatal porcine skin with 1.80 ±â€¯0.08 mg delivered over 24 h. Therefore, this initial study shows that microneedles do have the potential to prevent vitamin K deficiency bleeding. Future work will assess delivery of vitamin K in microneedle array in vivo.

Transdermal delivery of gentamicin using dissolving microneedle arrays for potential treatment of neonatal sepsis.

Neonatal infections are a leading cause of childhood mortality in low-resource settings. World Health Organization guidelines for outpatient treatment of possible serious bacterial infection (PSBI) in neonates and young infants when referral for hospital treatment is not feasible include intramuscular gentamicin (GEN) and oral amoxicillin. GEN is supplied as an aqueous solution of gentamicin sulphate in vials or ampoules and requires health care workers to be trained in dose calculation or selection of an appropriate dose based on the patient's weight band and to have access to safe injection supplies and appropriate sharps disposal. A simplified formulation, packaging, and delivery method to treat PSBI in low-resource settings could decrease user error and expand access to lifesaving outpatient antibiotic treatment for infants with severe infection during the neonatal period. We developed dissolving polymeric microneedles (MN) arrays to deliver GEN transdermally. MN arrays were produced from aqueous blends containing 30% (w/w) of GEN and two polymers approved by the US Food and Drug Administration: sodium hyaluronate and poly(vinylpyrrolidone). The arrays (19×19 needles and 500µm height) were mechanically strong and were able to penetrate a skin simulant to a depth of 378µm. The MN arrays were tested in vitro using a Franz Cell setup delivering approximately 4.45mg of GEN over 6h. Finally, three different doses (low, medium, and high) of GEN delivered by MN arrays were tested in an animal model. Maximum plasma levels of GEN were dose-dependent and ranged between 2 and 5µg/mL. The time required to reach these levels post-MN array application ranged between 1 and 6h. This work demonstrated the potential of dissolving MN arrays to deliver GEN transdermally at therapeutic levels in vivo.

Vial usage, device dead space, vaccine wastage, and dose accuracy of intradermal delivery devices for inactivated poliovirus vaccine (IPV).

INTRODUCTION: Intradermal delivery of a fractional dose of inactivated poliovirus vaccine (IPV) offers potential benefits compared to intramuscular (IM) delivery, including possible cost reductions and easing of IPV supply shortages. Objectives of this study were to assess intradermal delivery devices for dead space, wastage generated by the filling process, dose accuracy, and total number of doses that can be delivered per vial. METHODS: Devices tested included syringes with staked (fixed) needles (autodisable syringes and syringes used with intradermal adapters), a luer-slip needle and syringe, a mini-needle syringe, a hollow microneedle device, and disposable-syringe jet injectors with their associated filling adapters. Each device was used to withdraw 0.1-mL fractional doses from single-dose IM glass vials which were then ejected into a beaker. Both vial and device were weighed before and after filling and again after expulsion of liquid to record change in volume at each stage of the process. Data were used to calculate the number of doses that could potentially be obtained from multidose vials. RESULTS: Results show wide variability in dead space, dose accuracy, overall wastage, and total number of doses that can be obtained per vial among intradermal delivery devices. Syringes with staked needles had relatively low dead space and low overall wastage, and could achieve a greater number of doses per vial compared to syringes with a detachable luer-slip needle. Of the disposable-syringe jet injectors tested, one was comparable to syringes with staked needles. DISCUSSION: If intradermal delivery of IPV is introduced, selection of an intradermal delivery device can have a substantial impact on vaccine wasted during administration, and thus on the required quantity of vaccine that needs to be purchased. An ideal intradermal delivery device should be not only safe, reliable, accurate, and acceptable to users and vaccine recipients, but should also have low dead space, high dose accuracy, and low overall wastage to maximize the potential number of doses that can be withdrawn and delivered.

The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns.

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.

Ontogeny of Toll-like receptor mediated cytokine responses of human blood mononuclear cells.

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1ß varied by stimuli. Our data contradict the notion of a linear progression from an 'immature' neonatal to a 'mature' adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.

Identification of B cells through negative gating-An example of the MIFlowCyt standard applied.

Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell (1). Additional parameters may be analyzed through negative identification (i.e., through exclusion of particular stains or antibodies employed). In this report, we tested whether such negative-gating strategy would identify human B lymphocytes in innate immune phenotyping studies. To this end, B cells were identified as the negatively-stained subpopulation from the CD123 vs. CD11c plot of the CD14(neg-low), MHC II(high) human peripheral blood mononuclear cells. To test the specificity of this negative gating approach, we confirmed that negatively gated B cells indeed expressed CD19, the bona fide marker for human B cells. However, a small number of unidentified cells were contained in the negatively-gated B cells. Furthermore, a small percentage cells expressing markers used to identify monocytes and myeloid dendritic cells (mDC) coexpressed CD19. This identifies such negative B-cell gating approach as potentially problematic. When applied to the analysis of Toll-like receptors (TLR) stimulation experiments, we were however able to interpret the results, as B-cells respond to TLR stimulation in a qualitative different pattern as compared to monocytes and DC. This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) (2) and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt-compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data.

Neonatal innate TLR-mediated responses are distinct from those of adults.

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-alpha (and consequently induced less IFN-gamma), moderately less TNF-alpha, but as much or even more IL-1beta, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation.

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.

Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142.

Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.

ULBP4 is a novel ligand for human NKG2D.

The ULBPs are a family of MHC class I-related molecules. We have previously shown that ULBPs 1, 2, and 3 are functional ligands of the NKG2D/DAP10 receptor complex on human natural killer (NK) cells. Here, we describe a new member of the ULBP family, ULBP4, which contains predicted transmembrane and cytoplasmic domains, unlike the other ULBPs, which are GPI-linked proteins. Transduction of ULBP4 into EL4 cells confers the ability to bind recombinant NKG2D and mediates increased cytotoxic activity by human NK cells, consistent with the role of ULBPs as ligands for the NKG2D/DAP10 activating receptors. Tissue expression of ULBP4 differs from other members of the family, in that it is expressed predominantly in the skin.