Ivermectin Accelerates Circulating Nonstructural Protein 1 (NS1) Clearance in Adult Dengue Patients: A Combined Phase 2/3 Randomized Double-blinded Placebo Controlled Trial.

BACKGROUND: Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. METHODS: We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 µg/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 µg/kg ivermectin. RESULTS: The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9-84.0] hours vs 95.8 [95% CI 83.9-120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. CONCLUSIONS: A 3-day 1 daily dose of 400 µg/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin-Mediated Neutralization.

Flavivirus nonstructural protein 1 (NS1) is a unique secreted nonstructural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell associated. In this study, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties, including smaller and more heterogeneous sizes and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4, and C4-binding protein, as well as to a novel partner, mannose-binding lectin. Binding of NS1 to MBL protects DENV against mannose-binding lectin-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of infection.

An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies.

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples.

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device-termed Cygnus-with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-µl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58-0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63-0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.

Differential critical residues on the overlapped region of the non-structural protein-1 recognized by flavivirus and dengue virus cross-reactive monoclonal antibodies.

The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

Performance of a New Microfluidic Dengue NS1 Immuno-magnetic Agglutination Assay for the Rapid Diagnosis of Dengue Infection in Adults.

Dengue (DENV) infections are a public health concern worldwide and thus early diagnosis is important to ensure appropriate clinical management. The rapid diagnostic test (RDT) targets nonstructural protein 1 (NS1) detection and is the main tool used for diagnostic purpose. In this study, we evaluated the performance of a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay or IMA (ViroTrack Dengue Acute, BluSense Diagnostics, Copenhagen, Denmark). We studied 233 subjects confirmed to have DENV infection (by a real-time reverse transcriptase polymerase chain reaction) and 200 control samples were taken from patients with confirmed diagnoses of other febrile illnesses, in Thailand. Samples were tested using the NS1 antigen (Ag) detection methods: in-house NS1 Ag ELISA (ELISA), SD BIOLINE Dengue NS1 Ag RDT (ICT), and ViroTrack Dengue Acute (IMA). Sensitivities of these tests were 86.3%, 78.9%, and 85.5%, respectively. All tests showed high specificity (100%, 99%, and 97% for ELISA, ICT, and IMA, respectively). The sensitivities of both RDTs were affected by the low sensitivity to DENV-2 and DENV-4. NS1 Ag was detected in every patient on day 1 and day 2 after onset of illness by ELISA and IMA with a decline in detection rates over time after day 6 of illness. NS1 detection rate using ICT decreased from 100% on day 1 of illness to 98.6% on day 2 after onset of illness. By day 6, the detection rate was 45.9%. Thus, IMA performed better than ICT for early and rapid diagnosis of DENV infections in endemic countries.

Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection.

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

High performance dengue virus antigen-based serotyping-NS1-ELISA (plus): A simple alternative approach to identify dengue virus serotypes in acute dengue specimens.

Dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV). Four different serotypes (DENV1-4) co-circulate in dengue endemic areas. The viral RNA genome-based reverse-transcription PCR (RT-PCR) is the most widely used method to identify DENV serotypes in patient specimens. However, the non-structural protein 1 (NS1) antigen as a biomarker for DENV serotyping is an emerging alternative method. We modified the serotyping-NS1-enzyme linked immunosorbent assay (stNS1-ELISA) from the originally established assay which had limited sensitivity overall and poor specificity for the DENV2 serotype. Here, four biotinylated serotype-specific antibodies were applied, including an entirely new design for detection of DENV2. Prediction of the infecting serotype of retrospective acute-phase plasma from dengue patients revealed 100% concordance with the standard RT-PCR method for all four serotypes and 78% overall sensitivity (156/200). The sensitivity of DENV1 NS1 detection was greatly improved (from 62% to 90%) by the addition of a DENV1/DENV3 sub-complex antibody pair. Inclusive of five antibody pairs, the stNS1-ELISA (plus) method showed an overall increased sensitivity to 85.5% (171/200). With the same clinical specimens, a commercial NS1 rapid diagnostic test (NS1-RDT) showed 72% sensitivity (147/200), significantly lower than the stNS1-ELISA (plus) performance. In conclusion, the stNS1-ELISA (plus) is an improved method for prediction of DENV serotype and for overall sensitivity. It could be an alternative assay not only for early dengue diagnosis, but also for serotype identification especially in remote resource-limited dengue endemic areas.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy.

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects.

Production and characterization of anti-dengue capsid antibodies suggesting the N terminus region covering the first 20 amino acids of dengue virus capsid protein is predominantly immunogenic in mice.

We produced monoclonal and polyclonal antibodies to the capsid (C) protein of dengue serotype 2 virus (DV2 C). First, a maltose-binding protein fused to DV2 C protein (MBP-C) was overproduced in E. coli. The affinity-purified MBP-C protein was cleaved by factor Xa protease to obtain a recombinant DV2 C protein, which was then used for mouse immunizations. Two hybridoma cell lines producing anti-C Mabs as well as anti-C polyclonal antibody were successfully generated and characterized. Interestingly, all of the generated antibodies specifically recognized the first 20 amino acids of the DV2 C protein, as determined by peptide epitope mapping and via a recombinant DV2 C protein in which this region was deleted. The results suggested that this region is predominantly immunogenic in mice.

Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in Escherichia coli trxB and gor double mutant.

The dengue virus is currently the most important flavivirus causing human diseases in the tropical and subtropical regions of the world. The envelope protein domain III of dengue virus type 2 (D2EIII), which induces protective and neutralizing antibodies, was expressed as an N-terminal fusion to a hexa-histidine tag in Escherichia coli. The expression of recombinant D2EIII of 103 amino acids in the soluble form can be achieved using suitable host strains, such as Origami, at a low induction temperature of 18 degrees C. The enhanced production of the soluble protein could be attributed to the thioredoxin reductase (trxB) and glutathione reductase (gor) double mutations in the Origami genome. The soluble and refolded D2EIII proteins were recognized by different antibodies including human patient antiserum. The immunization of rats with soluble D2EIII protein elicited the production of antibodies that could recognize the D2EIII protein in the D2EIII precursor protein and in C-terminal truncated dengue envelope protein type 1-4. Thus, this protein production system is suitable for the production of authentic recombinant dengue proteins that may be used in the diagnosis of the dengue virus infection or in vaccine development.

The development of a novel serotyping-NS1-ELISA to identify serotypes of dengue virus.

BACKGROUND: Dengue virus (DENV), which causes mosquito-borne disease dengue hemorrhagic fever (DHF), consists of four serotypes co-circulating in endemic areas. Currently, DENV serotypes can be identified by laborious virus isolation followed by immunofluorescent assay and sophisticated RT-PCR. OBJECTIVE: To establish a new assay designated as "serotyping-NS1-ELISA" to detect the NS1 protein and to identify DENV serotypes simultaneously. STUDY DESIGN: The monoclonal antibodies (Mabs) against NS1 of each DENV serotype were produced and characterized for their serotype-specificity. To develop serotyping-NS1-ELISA, the selected serotype-specific anti-NS1 Mabs were applied to detect the NS1 antigen, which was previously captured by a flavivirus cross-reactive anti-NS1 Mab. Serotyping accuracy of the developed assay was validated with NS1 from DENV-infected cell culture supernatants and from well-characterized clinical specimens. RESULTS: Of 30 anti-NS1 Mabs, 1 serotype-specific anti-NS1 Mab to each DENV serotype was selected based on NS1 capture ELISA results for developing the serotyping-NS1-ELISA. Using DENV-infected cell culture supernatants for validation, the selected antibodies were shown to be capable of differentiating four DENV serotypes. When acute phase plasma from DENV-infected patients was used for validation, 65 out of 85 specimens (76.5% overall sensitivity) were positive to one of the four serotypes developed in our assay. Interestingly, identification of DENV serotypes by our serotyping-NS1-ELISA was 100% accurate for DENV1, 3 and 4 and 82.4% for DENV2 as compared with standard RT-PCR. Assay specificity was 100% (90/90). CONCLUSIONS: The developed serotyping-NS1-ELISA provides an alternative for simultaneous detection of DENV NS1 and identification of its serotype in acute patients' specimens. The assay would be applicable for dengue diagnosis and epidemiological studies.