David A. Muller

The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker.

The flavivirus nonstructural glycoprotein NS1 is an enigmatic protein whose structure and mechanistic function have remained somewhat elusive ever since it was first reported in 1970 as a viral antigen circulating in the sera of dengue-infected patients. All flavivirus NS1 genes share a high degree of homology, encoding a 352-amino-acid polypeptide that has a molecular weight of 46-55 kDa, depending on its glycosylation status. NS1 exists in multiple oligomeric forms and is found in different cellular locations: a cell membrane-bound form in association with virus-induced intracellular vesicular compartments, on the cell surface and as a soluble secreted hexameric lipoparticle. Intracellular NS1 co-localizes with dsRNA and other components of the viral replication complex and plays an essential cofactor role in replication. Although this makes NS1 an ideal target for inhibitor design, the precise nature of its cofactor function has yet to be elucidated. A plethora of potential interacting partners have been identified, particularly for the secreted form of NS1, with many being implicated in immune evasion strategies. Secreted and cell-surface-associated NS1 are highly immunogenic and both the proteins themselves and the antibodies they elicit have been implicated in the seemingly contradictory roles of protection and pathogenesis in the infected host. Finally, NS1 is also an important biomarker for early diagnosis of disease. In this article, we provide an overview of these somewhat disparate areas of research, drawing together the wealth of data generated over more than 40 years of study of this fascinating protein.

Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity

Dengue virus NS1 protein induces inflammatory responses via TLR4 and disrupts endothelial cell monolayer integrity. A leak in the dike Everyone knows how mosquitos can wreck an end-of-summer picnic. But in some climates, these pesky intruders persist and carry a variety of detrimental diseases—some with no preventative vaccines or targeted therapies. One such passenger is dengue virus (DENV), which infects up to 400 million people each year and comes in several serotypes (1 to 4) and disease presentations—from mild infection to severe disease and sometimes death. But to treat or prevent dengue requires that we have a more complete picture of the disease pathology. Now, Modhiran et al. and Beatty et al. describe the results of in vitro and in vivo experiments that point to circulating dengue virus non-structural protein 1 (NS1) and the innate immune Toll-like receptor 4 (TLR4) as a focus for basic scientists as well as vaccine and drug developers. DENV infection protects a patient from future reinfection with the same DENV serotype as well as producing temporary immune protection from severe dengue disease caused by a different DENV serotype. But unlike diamonds, this immune protection doesn’t last forever, and when the protected period passes, the patient becomes at increased risk of enhanced infection and progression to severe disease if he or she is infected with a second DENV serotype. This severe form of dengue infection is believed to result from immunopathogenic processes that induce cytokine storm and cause vascular leakage that leads to shock. Until now, no dengue viral proteins have been linked to vascular endothelium permeability (that is, vascular leakage). Beatty et al. show that inoculation of mice with DENV NS1 protein alone induces both vascular leak and secretion of inflammatory cytokines and that administration of NS1 with a sublethal dose of DENV2 leads to lethal vascular leak syndrome. In human endothelial cell monolayers in culture, NS1 from any of the four DENV serotypes triggered endothelial barrier permeability. NS1’s pathogenic effects were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1 (in vivo and in vitro), and immunization of mice with NS1 protected against lethal DENV2 challenge. In an independent study, Mondrian et al. explore the underlying mechanism of NS1’s effects. They show that highly purified NS1 acts as a pathogen-associated molecular pattern (PAMP) that activates mouse macrophages and human peripheral blood mononuclear cells (PBMCs) in culture via TLR4, resulting in release of inflammatory cytokines—an effect that was blocked by either a TLR4 antagonist or an anti-TLR4 antibody. Then, in an in vitro model of vascular leak, the authors found that NS1 fractured the integrity of endothelial cell monolayers through a TLR4-dependent pathway, a finding that was supported by the observation that a TLR4 antagonist quelled capillary leak in a mouse model of dengue virus infection. Together, these new findings highlight NS1 as an instigator of dengue-associated vascular leak and thus pinpoint a potential target for dengue drugs and component for dengue vaccines. Complications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP). Highly purified NS1 devoid of bacterial endotoxin activity directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via Toll-like receptor 4 (TLR4), leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. These results pinpoint NS1 as a viral toxin counterpart of the bacterial endotoxin lipopolysaccharide (LPS). Similar to the role of LPS in septic shock, NS1 might contribute to vascular leak in dengue patients, which highlights TLR4 antagonists as a possible therapeutic option.

Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis

The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

Surface modifications of microprojection arrays for improved biomarker capture in the skin of live mice

New technologies are needed to translate biomarker discovery research into simple, inexpensive, and effective molecular diagnostic assays for use by clinicians or patients to guide and monitor treatment. Microprojection arrays were recently introduced as tools which, when applied to the skin, penetrate into the dermal tissue, and capture specific circulating biomarkers. In our initial work on Microprojection arrays, carbodiimide chemistry was used to immobilize biomarker-specific probes for affinity capture in vivo using a mouse model. However, as the observed capture efficiencies were relatively low, with significant variation across the surface, here we investigated the surface modifications to (a) determine the source of the variability and (b) find ways of improving capture efficiency. We found the protein immobilization step accounted for almost all of the variability in surface uniformity. Varying the protein immobilization conditions following a standard carbodiimide activation process resulted in a reduction in overall variation 14-fold and an increase in captured biomarker amount ∼18-fold. In conclusion, we found that investigating and optimizing the surface chemistry of microprojection array devices led to drastic improvements in capturing biomarkers from skin fluid.

Surface modification and characterization of polycarbonate microdevices for capture of circulating biomarkers, both in vitro and in vivo.

Herein, we report the fabrication, characterization, and testing of a polymer microprojection array, for the direct and selective capture of circulating biomarkers from the skin of live mice. First, we modified polycarbonate wafers using an electrophilic aromatic substitution reaction with nitric acid to insert aromatic nitro-groups into the benzene rings, followed by treatment with sodium borohydride to reduce the nitro-groups to primary amines. Initial characterization by ultraviolet-visible (UV-vis) spectroscopy suggested that increasing acid concentration led to increased depth of material modification and that this was associated with decreased surface hardness and slight changes in surface roughness. Chemical analysis with X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance fourier transform infrared (ATR-FT-IR) spectroscopy showed nitrogen species present at the surface for all acid concentrations used, but subsurface nitrogen species were only observed at acid concentrations >35%. The nitrogen species were identified as a mixture of nitro, imine, and amine groups, and following reduction, there was sufficient amounts of primary amine groups for covalent attachment of a polyethylene glycol antifouling layer and protein capture probes, as determined by colorimetric and radiometric assays. Finally, the modification scheme was applied to polycarbonate microprojection arrays, and we show that these devices achieve flank skin penetration depths and biomarker yields comparable with our previously reported gold-coated silicon arrays, with very low nonspecific binding even in 10% mouse serum (in vitro) or directly in mouse skin (in vivo). This study is the first demonstration showing the potential utility of polymer microprojections in immunodiagnostics applications.

Clinical and Laboratory Diagnosis of Dengue Virus Infection

Infection with any of the 4 dengue virus serotypes results in a diverse range of symptoms, from mild undifferentiated fever to life-threatening hemorrhagic fever and shock. Given that dengue virus infection elicits such a broad range of clinical symptoms, early and accurate laboratory diagnosis is essential for appropriate patient management. Virus detection and serological conversion have been the main targets of diagnostic assessment for many years, however cross-reactivity of antibody responses among the flaviviruses has been a confounding issue in providing a differential diagnosis. Furthermore, there is no single, definitive diagnostic biomarker that is present across the entire period of patient presentation, particularly in those experiencing a secondary dengue infection. Nevertheless, the development and commercialization of point-of-care combination tests capable of detecting markers of infection present during different stages of infection (viral nonstructural protein 1 and immunoglobulin M) has greatly simplified laboratory-based dengue diagnosis. Despite these advances, significant challenges remain in the clinical management of dengue-infected patients, especially in the absence of reliable biomarkers that provide an effective prognostic indicator of severe disease progression. This review briefly summarizes some of the complexities and issues surrounding clinical dengue diagnosis and the laboratory diagnostic options currently available.

Capture of the circulating Plasmodium falciparum biomarker HRP2 in a multiplexed format, via a wearable skin patch

Herein we demonstrate the use of a wearable device that can selectively capture two distinct circulating protein biomarkers (recombinant P. falciparum rPfHRP2 and total IgG) from the intradermal fluid of live mice in situ, for subsequent detection in vitro. The device comprises a microprojection array that, when applied to the skin, penetrates the outer skin layers to interface directly with intradermal fluid. Because of the complexity of the biological fluid being sampled, we investigated the effects of solution conditions on the attachment of capture antibodies, to optimize the assay detection limit both in vitro and on live mice. For detection of the target antigen diluted in 20% serum, immobilization conditions favoring high antibody surface density (low pH, low ionic strength) resulted in 100-fold greater sensitivity in comparison to standard conditions, yielding a detection limit equivalent to the plate enzyme-linked immunosorbent assay (ELISA). We also show that blocking the device surface to reduce nonspecific adsorption of target analyte and host proteins does not significantly change sensitivity. After injecting mice with rPfHRP2 via the tail vein, we compared analyte levels in both plasma and skin biopsies (cross-sectional area same as the microprojection array), observing that skin samples contained the equivalent of ∼8 μL of analyte-containing plasma. We then applied the arrays to mice, showing that surfaces coated with a high density of antibodies captured a significant amount of the rPfHRP2 target while the standard surface showed no capture in comparison to the negative control. Next, we applied a triplex device to both control and rPfHRP2-treated mice, simultaneously capturing rPfHRP2 and total IgG (as a positive control for skin penetration) in comparison to a negative control device. We conclude that such devices can be used to capture clinically relevant, circulating protein biomarkers of infectious disease via the skin, with potential applications as a minimally invasive and lab-free biomarker detection platform.

A portable approach for the surveillance of dengue virus-infected mosquitoes.

Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses.

Riboflavin and ultraviolet light: impact on dengue virus infectivity

Dengue viruses (DENV 1‐4) are emerging across the world, and these viruses pose a risk to transfusion safety. Pathogen inactivation may be an alternative approach for managing the risk of DENV transfusion transmission. This study aimed to investigate the ability of riboflavin and UV light to inactivate DENV 1‐4 in platelet concentrates.

A Photothermal Spectrometer for Fast and Background-Free Detection of Individual Nanoparticles in Flow

Sensitive detection and quantification of individual plasmonic nanoparticles is critical in a range of applications in the biological, nanomaterials, and analytical sciences. Although a wide range of techniques can be applied to the analysis of immobilized particles, high-throughput analysis of nanoscale species in flow is surprisingly underdeveloped. To address this shortcoming, we present an ultrasensitive, background-free technique based on the photothermal effect and termed differential detection photothermal interferometry (DDPI). We show, both theoretically and experimentally, that DDPI can specifically extract either the phase or amplitude of a photothermal signal. We then quantitatively detect 10 and 20 nm diameter gold nanoparticles at femtomolar concentrations and at linear flow speeds of 10 mm/s. In the case of 50 nm gold particles, we operate at an even higher linear flow speed of 100 mm/s, corresponding to an analyzed volume of more than 1 nL/s. This allows quantification of particle content at attomolar to femtomolar concentrations and counting rates between 0.1 and 400 particles per second. Finally, we confirm that the signal follows the size-dependent variations predicted by Mie theory.