scholarly journals Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 726
Author(s):  
Nikole L. Warner ◽  
Kathryn M. Frietze
Keyword(s):  
Dengue Virus ◽  
Population Based ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Denv Serotypes ◽  
Conserved Regions ◽  
Virus Like Particle ◽  
Dependent Cell ◽  
Infected Cells ◽  
Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

scholarly journals Diterpenes/Diterpenoids and Their Derivatives as Potential Bioactive Leads against Dengue Virus: A Computational and Network Pharmacology Study

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6821
Author(s):  
Rasel Ahmed Khan ◽  
Rajib Hossain ◽  
Abolghasem Siyadatpanah ◽  
Khattab Al-Khafaji ◽  
Abul Bashar Ripon Khalipha ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Dengue Fever ◽  
Computational Study ◽  
Viral Proteins ◽  
Network Pharmacology ◽  
Binding Affinities ◽  
Ns1 Protein ◽  
Structural Protein ◽  
E Protein

Dengue fever is a dangerous infectious endemic disease that affects over 100 nations worldwide, from Africa to the Western Pacific, and is caused by the dengue virus, which is transmitted to humans by an insect bite of Aedes aegypti. Millions of citizens have died as a result of dengue fever and dengue hemorrhagic fever across the globe. Envelope (E), serine protease (NS3), RNA-directed RNA polymerase (NS5), and non-structural protein 1 (NS1) are mostly required for cell proliferation and survival. Some of the diterpenoids and their derivatives produced by nature possess anti-dengue viral properties. The goal of the computational study was to scrutinize the effectiveness of diterpenoids and their derivatives against dengue viral proteins through in silico study. Methods: molecular docking was performed to analyze the binding affinity of compounds against four viral proteins: the envelope (E) protein, the NS1 protein, the NS3 protein, and the NS5 protein. Results: among the selected drug candidates, triptolide, stevioside, alepterolic acid, sphaeropsidin A, methyl dodovisate A, andrographolide, caesalacetal, and pyrimethamine have demonstrated moderate to good binding affinities (−8.0 to −9.4 kcal/mol) toward the selected proteins: E protein, NS3, NS5, and NS1 whereas pyrimethamine exerts −7.5, −6.3, −7.8, and −6.6 kcal/mol with viral proteins, respectively. Interestingly, the binding affinities of these lead compounds were better than those of an FDA-approved anti-viral medication (pyrimethamine), which is underused in dengue fever. Conclusion: we can conclude that diterpenoids can be considered as a possible anti-dengue medication option. However, in vivo investigation is recommended to back up the conclusions of this study.

scholarly journals Association of dengue virus NS1 protein with lipid rafts

2008 ◽  
Vol 89 (10) ◽  
pp. 2492-2500 ◽  
Author(s):  
Sansanee Noisakran ◽  
Thanyaporn Dechtawewat ◽  
Panisadee Avirutnan ◽  
Taroh Kinoshita ◽  
Uamporn Siripanyaphinyo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dengue Virus ◽  
Lipid Rafts ◽  
Transmembrane Domain ◽  
Gradient Centrifugation ◽  
Ns1 Protein ◽  
Replication Complex ◽  
Infected Cells ◽  
A Minor ◽  
Triton X

During the replication of dengue virus, a viral non-structural glycoprotein, NS1, associates with the membrane on the cell surface and in the RNA replication complex. NS1 lacks a transmembrane domain, and the mechanism by which it associates with the membrane remains unclear. This study aimed to investigate whether membrane-bound NS1 is present in lipid rafts in dengue virus-infected cells. Double immunofluorescence staining of infected HEK-293T cells revealed that NS1 localized with raft-associated molecules, ganglioside GM1 and CD55, on the cell surface. In a flotation gradient centrifugation assay, a small proportion of NS1 in Triton X-100 cell lysate consistently co-fractionated with raft markers. Association of NS1 with lipid rafts was detected for all four dengue serotypes, as well as for Japanese encephalitis virus. Analysis of recombinant NS1 forms showed that glycosylated NS1 dimers stably expressed in HEK-293T cells without an additional C-terminal sequence, or with a heterologous transmembrane domain, failed to associate with lipid rafts. In contrast, glycosylphosphatidylinositol-linked recombinant NS1 exhibited a predilection for lipid rafts. These results indicate an association of a minor subpopulation of NS1 with lipid rafts during dengue virus infection and suggest that modification of NS1, possibly lipidation, is required for raft association.

scholarly journals Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method

Sensors ◽  
2021 ◽  
Vol 21 (23) ◽  
pp. 7809
Author(s):  
Kanaporn Poltep ◽  
Emi E. Nakayama ◽  
Tadahiro Sasaki ◽  
Takeshi Kurosu ◽  
Yoshiki Takashima ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Pcr Amplification ◽  
Denv Infection ◽  
Epidemiologic Studies ◽  
Cross Reaction ◽  
Structural Protein ◽  
Current Standard ◽  
Denv Serotypes ◽  
Detection Systems ◽  
Virus Serotype

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25–40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

scholarly journals Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis

Sensors ◽  
2018 ◽  
Vol 18 (8) ◽  
pp. 2641 ◽  
Author(s):  
Daniel Wasik ◽  
Ashok Mulchandani ◽  
Marylynn Yates
Keyword(s):  
Early Diagnosis ◽  
Dengue Virus ◽  
Dengue Infection ◽  
Denv Infection ◽  
Human Saliva ◽  
Diagnostic Methods ◽  
Label Free ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Blood Draw

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

scholarly journals Immunogenicity of Recombinant DNA Vaccine Encoding Non-Structural Protein-1 Dengue Virus Serotype-2 in Balb/c Mice

2021 ◽  
Vol 15 (2) ◽  
pp. 4
Author(s):  
Elitha Pulungan
Keyword(s):  
Immune Response ◽  
Dengue Virus ◽  
Dna Vaccine ◽  
Humoral Immune Response ◽  
Structural Proteins ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Humoral Immune ◽  
Virus Serotype ◽  
Positive Strand Rna

Background: Dengue Hemorrhagic Fever (DHF) is an infectious disease caused by the dengue virus (DENV) which spread widely in tropical and subtropical regions of the world. DENV is a single-positive strand RNA virus with a genome size of ± 11kb which encodes three structural proteins, seven non-structural proteins, and two untranslated regions (UTR). The non-structural protein-1 (NS1) of DENV is known to have important role in dengue pathogenesis also promising to be developed as dengue vaccine. Lately, novel vaccine approach by DNA immunization have given new perspective for a safe, stable, and immunogenic vaccine platform. Previously, we have successfully construct DNA vaccine encoding NS1 protein of DENV2 (pUNS1) which express recombinant NS1 protein in-vitro. Thus, in this current study the ability of pUNS1 to induce humoral immune response will be further analyzed by in mice immunization. Methods: Sixteen BALB/c mice aged of 4 weeks were immunized 3 times with 100 µg of pUNS1 or pUMVC4a on 2 week time interval. Blood sampling was carried out just before immunization and termination was done 2 week after last immunization. Titer from individual mice sera against DENV-2 were measure with in-house ELISA. Results: IgG against NS1 protein of DENV2 titer from mice group immunized with recombinant pUNS1 shown high ELISA absorbancies, 5 times higher than pUMVC4a group. This result suggest the ability of pUNS1 to induce humoral immune response against NS1 DENV-2 in-vivo. Conclusion: Recombinant pUNS1 can induce humoral immune response in mice.

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

2021 ◽  
Vol 15 (2) ◽  
pp. e0009065
Author(s):  
Tanapan Prommool ◽  
Pongpawan Sethanant ◽  
Narodom Phaenthaisong ◽  
Nattaya Tangthawornchaikul ◽  
Adisak Songjaeng ◽  
...  
Keyword(s):  
Dengue Virus ◽  
High Performance ◽  
Hemorrhagic Fever ◽  
Virus Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Structural Protein ◽  
Rt Pcr ◽  
Denv Serotypes ◽  
Reverse Transcription Pcr ◽  
Endemic Areas

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.

scholarly journals Computational antigenic epitope prediction of clinical Indonesian Dengue virus NS1 protein

2021 ◽  
Vol 948 (1) ◽  
pp. 012080
Author(s):  
S Pambudi ◽  
D Irawan ◽  
A Danny ◽  
T Widayanti ◽  
Tarwadi
Keyword(s):  
T Cell ◽  
Dengue Virus ◽  
B Cell ◽  
Dengue Infection ◽  
Epitope Prediction ◽  
Antigenic Epitope ◽  
Ns1 Protein ◽  
Mhc I ◽  
B Cell Epitopes ◽  
Denv Serotypes

Abstract The identification of human Non-Structural-1 (NS1) protein epitopes will help us better understand Dengue virus (DENV) immunopathogenesis. In this study, several online and offline bioinformatic prediction tools were exploited to predict and analyze T-cell and B-cell epitopes of DENV NS1 consensus sequences originated from Indonesian clinical isolates. We identified a potential peptide at NS1155--163 (VEDYGFGIF) which interact with MHC-I allele HLA-B*40:01 and showed high binding affinity (IC50) scores ranging between 63.8 nM to 183.9 nM for all Indonesian DENV serotypes. Furthermore, we have succeeded identified a region at the C-terminal of Indonesian DENV NS1 protein between 325--344 as part of discontinuous antigenic epitope which conserved for all serotypes. Our analyses showed this region could induce strong and persistent antibody against all DENV serotypes by interacting with MHC-I molecule and also recognized by B-cell receptor. The identification of DENV NS1 T-cell and B-cell epitopes may help in the development of a new vaccine, drug discovery, and diagnostic system to help eradicate dengue infection.

scholarly journals A New Approach in DENV Diagnostic based on Linear Peptides Obtained by Phage Display

2021 ◽  
Author(s):  
Mirna Burciaga-Flores ◽  
Tanya A. Camacho-Villegas ◽  
Pavel H. Lugo-Fabres ◽  
Abel Gutiérrez-Ortega ◽  
José Esteban Muñoz-Medina ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Phage Display ◽  
Early Stage ◽  
In Silico Analysis ◽  
Cross Reactivity ◽  
Diagnostic Tools ◽  
Correct Identification ◽  
Binding Modes ◽  
Ns1 Protein ◽  
Denv Serotypes

Abstract Dengue is a viral disease caused by any of the four distinct dengue virus (DENV) serotypes that circulate in many parts of the world. DENV now co-circulates with Zika and Chikungunya viruses (ZIKV and CHIKV) in many regions of the Americas. Having this in mind, plus the fact that DENV clinical diagnosis persists as a difficult task, due to the similarity in symptoms, as well as false-positive results by the cross-reactivity of the IgG and IgM against these three viruses, correct identification of DENV at an early stage of the disease is essential to minimise transmission and prevent potentially devastating sequelae. Here, by phage display, we isolate specific peptides for dengue virus NS1 protein. The specificity of the linear peptides as diagnostic tools for DENV NS1 protein in sera samples was investigated, and the selected peptides showed the ability to recognise DENV, and no cross-reactivity was shown. Moreover, in silico analysis was performed to assess the possible binding modes of these peptides to DENV-NS1, using a molecular docking approach. These peptides are suitable for use in an ELISA assay for dengue virus detection in human serum and the possibility to adapt these peptides to PoC platforms.

scholarly journals Increased Plasma Heparanase Activity and Endothelial Glycocalyx Degradation in Dengue Patients Is Associated With Plasma Leakage

2021 ◽  
Vol 12 ◽  
Author(s):  
Baranca Buijsers ◽  
Fadel Muhammad Garishah ◽  
Silvita Fitri Riswari ◽  
Rosalie M. van Ast ◽  
Setyo Gundi Pramudo ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Heparan Sulfate ◽  
Ex Vivo ◽  
Endothelial Glycocalyx ◽  
Severe Dengue ◽  
Structural Protein ◽  
Dengue Virus Infection ◽  
Activated Platelets ◽  
Plasma Leakage

BackgroundEndothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection.MethodsHeparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo.ResultsHeparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets.ConclusionTaken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase.

scholarly journals Horseradish peroxidase-labeled rabbit anti-non-structural protein 1 of dengue virus-2 for the diagnosis of dengue virus infections

2019 ◽  
Vol 28 (2) ◽  
pp. 103-9
Author(s):  
Evy Suryani Arodes ◽  
Beti Ernawati Dewi ◽  
Tjahjani Mirawati Sudiro
Keyword(s):  
Early Diagnosis ◽  
Horseradish Peroxidase ◽  
Dengue Virus ◽  
Periodate Oxidation ◽  
Denv Infection ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Ns1 Antigen

BACKGROUND Early diagnosis of dengue virus (DENV) infection is essential for patient management and disease control. Detection of the antigen non-structural protein 1 (NS1) has been proven to provide early diagnosis of DENV infection. Thus, commercial NS1 antigen detection assays have been increasingly used and are becoming thetool of choice among clinicians to confirm DENV infection in Indonesia. METHODS To obtain anti-NS1 DENV antibody, NS1 protein (90 µg/ml) from the collection of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia was injected into a rabbit. The anti-NS1 antibody from the rabbit was then labeled with horseradish peroxidase (HRP) using the periodate oxidation method. Sera were tested by enzyme-linked immunosorbent assay (ELISA) to detect NS1 from DENV-infected patients. RESULTS Serially diluted antibody labeled with HRP tested using the direct ELISA method showed the highest absorbance value at a 1:100 dilution (Mean [SD] = 1.35 [0.35]); even at a dilution as high as 1:3,200 (0.22 [0.15]), antibody labeled with HRP was able to detect the NS1 protein, although the absorbance value did not differ greatly from that of the negative control (0.13 [0.01]). CONCLUSIONS In an attempt to develop an NS1-based diagnostic test, polyclonal anti-NS1 DENV antibody was successfully produced as a diagnostic assay to determine the presence of DENV NS1 antigen in patients’ sera.

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