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 Toll-like receptors (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, conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively), produced less IL-12p70 and IFN-α (and consequently induced less IFN-γ), moderately less TNF-α, but as much or even more IL-1β, 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.

f 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 ϳ99% 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 nextgeneration sequencing.

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-1β 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.

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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.

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 500 μm height) were mechanically strong and were able to penetrate a skin simulant to a depth of 378 μm. The MN arrays were tested in vitro using a Franz Cell setup delivering approximately 4.45 mg of GEN over 6 h. 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 5 μg/mL. The time required to reach these levels post-MN array application ranged between 1 and 6 h. This work demonstrated the potential of dissolving MN arrays to deliver GEN transdermally at therapeutic levels in vivo.

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.