Efficacy and immunogenicity following dengue virus-1 human challenge after a tetravalent prime-boost dengue vaccine regimen: an open-label, phase 1 trial.

BACKGROUND: Dengue human infection models (DHIMs) are important tools to down-select dengue vaccine candidates and establish tetravalent efficacy before advanced clinical field trials. We aimed to provide data for the safety and immunogenicity of DHIM and evaluate dengue vaccine efficacy. METHODS: We performed an open-label, phase 1 trial at the University of Maryland (Baltimore, MD, USA). Eligible participants were healthy individuals aged 18-50 years who either previously received a tetravalent dengue purified inactivated vaccine prime followed by a live-attenuated vaccine boost (ie, the vaccinee group), or were unvaccinated flavivirus-naive participants (ie, the control group). Participants in the vaccinee group with detectable pre-challenge dengue virus-1 neutralising antibody titres and flavivirus-naive participants in the control group were inoculated with dengue virus-1 strain 45AZ5 in the deltoid region, 27-65 months following booster dosing. These participants were followed-up from days 4-16 following dengue virus-1 live virus human challenge, with daily real-time quantitative PCR specific to dengue virus-1 RNA detection, and dengue virus-1 solicited local and systemic adverse events were recorded. The primary outcomes were safety (ie, solicited local and systemic adverse events) and vaccine efficacy (ie, dengue virus-1 RNAaemia) following dengue challenge. This study is registered with ClinicalTrials.gov, number NCT04786457. FINDINGS: In January 2021, ten eligible participants were enrolled; of whom, six (60%) were in the vaccinee group and four (40%) were in the control group. Daily quantitative PCR detected dengue virus-1 RNA in nine (90%) of ten participants (five [83%] of six in the vaccinee group and all four [100%] in the control group). The mean onset of RNAaemia occurred on day 5 (SD 1·0) in the vaccinee group versus day 8 (1·5) in the control group (95% CI 1·1-4·9; p=0·007), with a trend towards reduced RNAaemia duration in the vaccinee group compared with the control group (8·2 days vs 10·5 days; 95% CI -0·08 to 4·68; p=0·056). Mild-to-moderate symptoms (nine [90%] of ten), leukopenia (eight [89%] of nine), and elevated aminotransferases (seven [78%] of nine) were commonly observed. Severe adverse events were detected only in the vaccinee group (fever ≥38·9°C in three [50%] of six, headache in one [17%], and transient grade 4 aspartate aminotransferase elevation in one [17%]). No deaths were reported. INTERPRETATION: Participants who had tetravalent dengue purified inactivated vaccine prime and live-attenuated vaccine boost were unprotected against dengue virus-1 infection and further showed increased clinical, immunological, and transcriptomic evidence for inflammation potentially mediated by pre-existing infection-enhancing antibodies. This study highlights the impact of small cohort, human challenge models studying dengue pathogenesis and downstream vaccine development. FUNDING: Military Infectious Disease Research Program and Medical Technology Enterprise Consortium and Advanced Technology International.

CLIPB4 Is a Central Node in the Protease Network that Regulates Humoral Immunity in Anopheles gambiae Mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPB4 is a central node in the protease network that regulates humoral immunity in Anopheles gambiae mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Optical biosensing of markers of mucosal inflammation.

We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.

Measuring stimulation and inhibition of intracellular nitric oxide production in SIM-A9 microglia using microfluidic single-cell analysis.

Chronic neuroinflammation has long been considered to be a central factor in accelerating the progression of neurodegenerative diseases such as Alzheimer's diseases, Parkinson's disease and chronic traumatic encephalopathy. Under pathological conditions microglia produce inflammatory signaling molecules, such as nitric oxide (NO), that can damage DNA and proteins and ultimately induce neuronal apoptosis. One strategy for treating neurodegenerative diseases is to specifically target NO production through inhibition of inducible nitric oxide synthase (iNOS). However, accurately measuring changes in microglial NO production in response to potential therapeutics is challenging due to NO's short half-life and microglial heterogeneity. In this paper we report the application of a microfluidic device for the high-throughput measurement of intracellular NO in SIM-A9 microglial cells. NO production was measured in response to treatment with lipopolysaccharides (LPS) and interferon gamma (IFN-γ) with and without a potent iNOS inhibitor (1400 W dihydrochloride). Cells were labeled with a fluorogenic NO probe, 4-amino-5-methylamino-2',7'-difluorofluoescein diacetate (DAF-FM DA), and 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. Separation and quantitation of intracellular NO was achieved using microchip electrophoresis and laser induced fluorescence detection (LIF). Statistical analysis suggests that the populations fit a lognormal distribution and are better represented by their geometric mean values. Comparison of the geometric means indicated a 1.6-fold increase in NO production between untreated and stimulated cells and a decrease by a factor of approximately 0.5 comparing stimulated and inhibited cells. Additionally, we report experimental data demonstrating the improvement in the sensitivity of our integrated optical fiber-based detection system through the use of refractive index matching gel.

Microelectrophoretic single-cell measurements with microfluidic devices.

Here we describe in detail the design, fabrication and operation of our automated high-throughput single cell microchip electrophoresis device with laser induced fluorescence detection. Our device features on-board integrated peristaltic pumps that generate flow directly within the microfluidic channels. Additionally, we have incorporated an optical fiber bridge that enables simultaneous fluorescence detection at two points of interest within the device without the need for additional optical components or detectors. The second detection spot is used to detect the intact cell immediately prior to lysis giving a signal at t=0s for each single-cell electropherogram. We can also use this signal to measure the absolute migration time of the separated analytes to confidently determine the identity of each peak. Finally, we demonstrate the application of our device for the measurement of intracellular nitric oxide (NO) levels in T-lymphocytes. Changes in NO levels within cells is associated with a number of chronic diseases including neurodegenerative, cardiovascular and cancers. We show that our system is capable of measuring NO levels under the following conditions: native, lipopolysaccharide stimulation, and inhibition of inducible nitric oxide synthase. It is our hope that the information and procedures described in this chapter may enable others to use or adapt our system for other analyses at the single cell level.

Fabrication of Glass Microfluidic Devices.

This chapter provides step-by-step procedures for the fabrication of glass-based microfluidic devices. These procedures include device design, photomask generation, photolithography, channel etching, and high-temperature bonding.

Early detection of pancreatic cancers in liquid biopsies by ultrasensitive fluorescence nanobiosensors.

Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and - 9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses.

Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

AC dielectrophoretic manipulation and electroporation of vaccinia virus using carbon nanoelectrode arrays.

This paper reports the capture and detection of vaccinia virus particles based on AC dielectrophoresis (DEP) and electrochemical impedance measurements employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in a "points-and-lid" configuration. The nano-DEP device was fabricated by bonding two SU-8 covered electrodes patterned using photolithography. The bottom electrode contains a 200 × 200 µm2 active region on a randomly distributed NEA and the top electrode contains a microfluidic channel in SU-8 spin-coated on ITO to guide the flow of the virus solution. The real-time impedance change was measured during DEP capture and validated with fluorescence microscopy measurements. The NEA was able to capture virus particles with a rather low AC voltage (∼8.0 V peak-to-peak) at 1.0 kHz frequency as the particles were passed through the fluidic channel at high flow velocities (up to 8.0 mm/s). A concentration detection limit as low as ∼2.58 × 103 particles/mL was obtained via impedance measurements after only 54 sec of DEP capture. At the low AC frequencies (50.0 Hz or less), the high electric field at the exposed VACNF tips induced electroporation of the DEP-captured virus particles, which was validated by fluorescence emission from the dyes staining lipophilic membrane and internal nucleic acid, respectively. This study suggests the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic viruses, with a potential of generating electroporation to the captured the virus particles for further biochemical study.

Out-of-plane integration of a multimode optical fiber for single particle/cell detection at multiple points on a microfluidic device with applications to particle/cell counting, velocimetry, size discrimination and the analysis of single cell lysate injections.

In this paper a single particle/cell-tracking microfluidic device that integrates an out-of-plane multimode optical fiber (OP-MMF) is reported. This OP-MMF is used to generate three excitation light-lines and three detection spots using only one excitation source and one detector. It takes advantage of an optical tunneling mode to create two excitation lines in a microfluidic channel emanating from a single fiber end. This method was used to accurately count particles/cells and perform velocity measurements and size discrimination. The velocity and size distributions of 5, 7, and 10 µm fluorescently labeled polystyrene beads were determined using the OP-MMF. Additionally, this method was used to analyze cell lysates with the third excitation line in the separation channel. The OP-MMF setup accurately detected an intact cell twice ∼2 mm prior to lysis, determined its velocity, and detected the injected cell lysate 3 mm downstream of the injection point in the separation channel. Using this setup, the velocity of cells entering the lysis intersection and the absolute migration times of fluorescently labeled analytes injected into the separation channel were determined in an automated fashion. This method enabled us to determine a lysing/injection efficiency coefficient (K) using signals from the injected lysate signal and from the intact cell before lysing. K provided a reliable measurement of the amount of cell lysate that was injected into the separation channel. The approach reported here could be used in the future to track particles, cells or droplets in a variety of existing microfluidic devices without the need for multiplexed masks, layers, bulky optical elements or complex optical designs.

Integrating Optical Fiber Bridges in Microfluidic Devices to Create Multiple Excitation/Detection Points for Single Cell Analysis.

A microfluidic device is reported that employs an out-of-plane optical fiber bridge to generate two excitation and two detection spots in a microfluidic channel using only one excitation source and one detector. This fiber optic bridge was integrated into a single cell analysis device to detect an intact cell just prior to lysis and the injected lysate 2, 5, 10, or 15 mm downstream of the injection point. Using this setup the absolute migration times for analytes from cells stochastically entering the lysis intersection could be determined for the first time in an automated fashion. This allowed the evaluation of several separation parameters, including analyte band velocity, migration time drift, diffusion coefficient, injection plug length, separation efficiency (N), and plate height (H), which previously could only be estimated. To demonstrate the utility of this system, a peptide substrate for protein kinase B (PKB) was designed, synthesized, and loaded into T-lymphocytes in order to measure PKB activity in individual cells. The optical fiber bridge is easy to implement, inexpensive, and flexible in terms of changing the distances between the two detection points.

High-throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps.

The ability to accurately control fluid transport in microfluidic devices is key for developing high-throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time-consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low-cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T-Lymphocyte cells loaded with Oregon green and 6-carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single-cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady-state population of immortalized cells.

Effect of Potassium Citrate on Calcium Phosphate Stones in a Model of Hypercalciuria.

Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation.

Integration of a nanostructured dielectrophoretic device and a surface-enhanced Raman probe for highly sensitive rapid bacteria detection.

This work reports a synergistic approach to the concentration, detection and kinetic monitoring of pathogens through the integration of nanostructured dielectrophoresis (DEP) with nanotag-labelled Surface Enhanced Raman Spectroscopy (SERS). A nanoelectrode array made of embedded Vertically Aligned Carbon Nanofibers (VACNFs) at the bottom of a microfluidic chip was used to effectively capture and concentrate nanotag-labelled E. coli DHα5 cells into a 200 µm × 200 µm area on which a Raman laser probe was focused. The SERS nanotags were based on iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of ∼50 nm size, which were coated with a QSY21 Raman reporter and attached to E. coli through specific immunochemistry. The combination of the greatly enhanced Raman signal by the SERS nanotags and the effective DEP concentration significantly improved the detection limit and speed. The SERS signal was measured with both a confocal Raman microscope and a portable Raman probe during DEP capture, and was fully validated with fluorescence microscopy measurements under all DEP conditions. The SERS measurements were sensitive enough to detect a single bacterium. A concentration detection limit as low as 210 cfu ml(-1) using a portable Raman system was obtained with a DEP capture time of only ∼50 s. These results demonstrate the potential to develop a compact portable system for rapid and highly sensitive detection of specific pathogens. This system is reusable, requires minimum sample preparation, and is amenable to field applications.

Persistence of 1,25D-induced hypercalciuria in alendronate-treated genetic hypercalciuric stone-forming rats fed a low-calcium diet.

Genetic hypercalciuric stone-forming (GHS) rats demonstrate increased intestinal Ca absorption, increased bone resorption, and reduced renal tubular Ca reabsorption leading to hypercalciuria and all form kidney stones. GHS have increased vitamin D receptors (VDR) at these sites of Ca transport. Injection of 1,25(OH)2D3 (1,25D) leads to a greater increase in urine (u)Ca in GHS than in control Sprague-Dawley (SD), possibly due to the additional VDR. In GHS the increased uCa persists on a low-Ca diet (LCD) suggesting enhanced bone resorption. We tested the hypothesis that LCD, coupled to inhibition of bone resorption by alendronate (alen), would eliminate the enhanced 1,25D-induced hypercalciuria in GHS. SD and GHS were fed LCD and half were injected daily with 1,25D. After 8 days all were also given alen until euthanasia at day 16. At 8 days, 1,25D increased uCa in SD and to a greater extent in GHS. At 16 days, alen eliminated the 1,25D-induced increase in uCa in SD. However, in GHS alen decreased, but did not eliminate, the 1,25D-induced hypercalciuria, suggesting maximal alen cannot completely prevent the 1,25D-induced bone resorption in GHS, perhaps due to increased VDR. There was no consistent effect on mRNA expression of renal transcellular or paracellular Ca transporters. Urine CaP and CaOx supersaturation (SS) increased with 1,25D alone in both SD and GHS. Alen eliminated the increase in CaP SS in SD but not in GHS. If these results are confirmed in humans with IH, the use of bisphosphonates, such as alen, may not prevent the decreased bone density observed in these patients.

1,25(OH)2D3 induces a mineralization defect and loss of bone mineral density in genetic hypercalciuric stone-forming rats.

Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (u) calcium (Ca) excretion, demonstrate increased intestinal Ca absorption, increased bone Ca resorption, and reduced renal Ca reabsorption, all leading to elevated uCa compared to the parental Sprague-Dawley (SD) rats. GHS rats have increased numbers of vitamin D receptors (VDRs) at each site, with normal levels of 1,25(OH)2D3 (1,25D), suggesting their VDR is undersaturated with 1,25D. We have shown that 1,25D induces a greater increase in uCa in GHS than SD rats. To examine the effect of the increased VDR on the osseous response to 1,25D, we fed GHS and SD rats an ample Ca diet and injected either 1,25D [low dose (LD) 12.5 or high dose (HD) 25 ng/100 g body weight/day] or vehicle (veh) daily for 16 days. Femoral areal bone mineral density (aBMD, by DEXA) was decreased in GHS+LD and GHS+HD relative to GHS+veh, while there was no effect on SD. Vertebral aBMD was lower in GHS compared to SD and further decreased in GHS+HD. Both femoral and L6 vertebral volumetric BMD (by µCT) were lower in GHS and further reduced by HD. Histomorphometry indicated a decreased osteoclast number in GHS+HD compared to GHS+veh or SD+HD. In tibiae, GHS+HD trabecular thickness and number increased, with a 12-fold increase in osteoid volume but only a threefold increase in bone volume. Bone formation rate was decreased in GHS+HD relative to GHS+veh, confirming the mineralization defect. The loss of BMD and the mineralization defect in GHS rats contribute to increased hypercalciuria; if these effects persist, they would result in decreased bone strength, making these bones more fracture-prone. The enhanced effect of 1,25D in GHS rats indicates that the increased VDRs are biologically active.