Anti-dengue NS1 Research Articles

Epidemiological investigation of dengue fever outbreak and its socioeconomic determinants in Banadir region, Somalia

BackgroundDengue has become an alarming global problem and is endemic in many countries, particularly in tropical and subtropical countries. The aim of this study was to investigate dengue fever outbreak in Banadir Region, Somalia, to understand the risk factors (time, place, personal characteristics).MethodsA descriptive cross-sectional study was undertaken to determine the levels of circulating anti-dengue virus antibodies and DENV NS1 antigen among Banadir Region residents, while a questionnaire survey was conducted to understand the clinical and demographic characteristics of the patients.ResultsA total of 735 febrile patients were studied, with 55.6% men and 44.3% women. The majority of the participants were children aged 14 years and younger. Among them, 10.8% tested positive for IgM antibodies against dengue virus (DENV), while the prevalence of DENV NS1 antigen was 11.8%. Fever and myalgia were the most common symptoms observed in the DENV-positive patients.ConclusionsA dengue fever outbreak has been confirmed in Banadir region, Somalia. This study provides information on the most affected districts and identifies risk factors contributing to DF outbreaks. The study recommends improving outbreak readiness and response, particularly in surveillance and laboratory diagnostics, by fostering intersectoral collaboration and establishing regulatory frameworks for financial and operational participation.

A systematic review and meta-analysis on the accuracy of rapid immunochromatographic tests for dengue diagnosis.

Rapid immunochromatographic tests are frequently used to diagnose dengue due to their easy use, low cost, and fast response. A high level of accuracy is essential for rapid diagnostic tests to support their large-scale use. Thus, this systematic review aims to evaluate the accuracy of rapid dengue diagnostic tests. The investigation was run through the following databases: LILACS, Medline (Pubmed), CRD, The Cochrane Library, Trip Medical Database, and Google Scholar. To solve difficulties, two independent reviewers performed document screening and selection. ELISA assay was adopted as a reference test because of several methodologic advantages. Seventeen articles were included accordingly, reckoning 6837 participating individuals. The receiver operating characteristic (ROC) and Forest Plot were conducted to evaluate the sensitivity and specificity for each analyzed parameter (anti-dengue IgM, IgG, and NS1 antigen). The risk of bias and quality of evidence were assessed as moderate using QUADAS-2 and Grading of Recommendations Assessment, Development, and Evaluation (GRADE), respectively. The sensitivity of IgM concerning the studied tests ranged from 13.8 to 90%, while that of NS1 ranged from 14.7 to 100% (95% CI). The antibodies with NS1 presented increased sensitivity; pooled data show that the association of the three analytes bestows the best result, with a combined sensitivity of 90% (CI 95%: 87-92%) and a pooled specificity of 89% (CI 95%: 87-92%). Thus, the present review provides relevant knowledge for decision-making between available rapid diagnostic tests.

Characterization of Human Anti-Dengue NS1 Monoclonal Antibodies Derived from Patients Infected with DENV-2.

Mouse antibodies specific to dengue NS1 have been widely investigated for their cross-reactivity with several human biomolecules. This is the first study demonstrating the cross-reactivity of human monoclonal antibodies (HuMAbs) specific to dengue NS1 isolated from patients infected with dengue virus serotype-2 (DENV-2). Nine anti-NS1 HuMAbs, which were mainly derived from patients in convalescent-phase after secondary infection of DENV-2, were characterized. Their cross-reactivity with plasminogen, thrombin, and endothelial cells was investigated, following which plasmin-formation assays were performed. All anti-NS1 HuMAbs exhibited cross-reactivity with human plasminogen (Plg), but not with thrombin or endothelial cells. Moreover, all HuMAbs exhibiting cross-reactivity with Plg converted Plg to plasmin in the plasmin-formation assay. These results suggest the implications and drawbacks of using anti-NS1 antibodies in immunotherapy.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion

Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells.To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas.VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays.Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs.A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

Epidemiology and immuno-molecular status of Nepal dengue outbreak

Background: The emergence and circulation of dengue infection have become one of the major public health concerns in the world with its high prevalence in the sub-tropical and tropical countries. Hundreds of Dengue cases have been reported annually and the shift of serotypes in every year has raised the concern of public health in Nepal. The detection of dengue virus infections and circulation of different serotypes in different areas has a great role for the clinical management of patients, surveillance, and clinical trial assessments in the days to come. Methods and materials: Clinically suspected dengue cases (n = 240) were subjected to sero analysis for anti-dengue IgM, IgG antibody and NS1 antigen followed by IgG end-point ELISA. Viral RNA extraction was performed for all samples using the Qiagen kit. cDNA was prepared, and RT-PCR was performed using D1 and DencomR2 primers. Dengue Serotyping was done for PCR positive samples using gene-specific primers. Results: Out of 240 dengue suspected cases, male (n = 153/63.75%), female (n = 87/36.25%) and children below 10 years (6.25%) were diagnosed as positive by ELISA. NS1 (59.16%), IgM (33%) and IgG (7.9%) were detected by ELISA, however, only 14 samples were positive alone to each test. Most of the cases were found to be of primary infection. Antibody level for each serum was calculated and categorized as high (43228.70), medium and low (953.53). Only 35.83% of the suspected cases were confirmed positive for dengue virus by PCR and were of Dengue Serotype-1 in the year 2016 in Nepal. Conclusion: The single test alone could not be confirmatory as only 35.83% of the clinically suspected cases were confirmed for Dengue virus infection by PCR. This implies that confirmatory tests like PCR should be coupled with the ELISA results for the proper diagnosis and management of the disease.

Dengue NS1 detection in pediatric serum using microfluidic paper-based analytical devices.

The diagnosis of dengue infection is still a critical factor determining success in the clinical management and treatment of patients. Here, the development of microfluidic paper-based analytical devices (μPADs) utilizing a sandwich immunoassay on wax patterned paper functionalized with anti-dengue NS1 monoclonal antibodies for point-of-care detection of dengue NS1 (DEN-NS1-PAD) is reported. Various assay conditions, including the length of the channel and diluent, were optimized, and the response detected by the naked eye and digitized images within 20-30min. The DEN-NS1-PAD was successfully tested in the field for detecting dengue NS1 in buffer, cell culture media, and human serum. The limit of detection (LoD) of the DEN-NS1-PAD obtained with the naked eye, scanner, and a smartphone camera was 200, 46.7, and 74.8ngmL-1, respectively. The repeatability, reproducibility, and stability of the DEN-NS1-PAD were also evaluated. High true specificity and sensitivity in the serum of pediatric patients were observed. These evaluation results confirm that the DEN-NS1-PAD can potentially be used in point-of-care dengue diagnostics, which can significantly impact on the spreading of mosquito-borne diseases, which are likely to become more prevalent with the effects of global warming. Graphical Abstract.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion.

Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

A cross sectional study of dengue virus infection in febrile patients presumptively diagnosed of malaria in Maiduguri and Jos plateau, Nigeria.

In Nigeria, where malaria is endemic, greater than 70% of febrile illnesses are treated presumptively as malaria, often without a laboratory evaluation for other possible causes of fever. This cross-sectional study evaluated the presence of dengue virus infection in febrile patients, presumptively diagnosed of malaria infections in the clinic. Blood samples were collected from 529 febrile patients (246 in Jos and 283 in Maiduguri) attending the general outpatient clinics of the Jos University Teaching Hospital (JUTH) and the University of Maiduguri Teaching Hospital (UMTH) and tested for anti-dengue immunoglobulin M (IgM) and immunoglobulin G (IgG), as well as anti-non-structural protein (NS1) by ELISA. The samples were also evaluated for presence of P. falciparum malaria parasites by microscopic examination of Giemsa-stained blood smears. The prevalence of confirmed, highly suggestive and probable dengue virus infections categorized in relation to duration of illness since onset of fever were 2.3%, 5.5% and 1.5% respectively, while the prevalence of anti-flavivirus IgG and IgM seropositivity was 11.7%. In a total of 117 (22.1%) patients (32 in Jos, 85 in Maiduguri), malaria parasites were detected by blood smear microscopy, out of which 7 (6%) also had a positively confirmed, highly suggestive or probable dengue test result. Although the high cross-reactivity of anti-flavivirus antibodies should be taken into account in the interpretation of the seroprevalence data, our findings suggest a significant presence of dengue virus in this environment, some of which may otherwise be misdiagnosed as malaria. These findings are strong enough to recommend serological screening for anti-dengue virus titer and NS1 antigen for all febrile patients, as part of fever diagnostic protocols in tropical regions. Given the prevalence of dengue virus infections, there is also a need for a dengue control program and public education to prevent outbreaks and occurrence of severe dengue complications.

Bio-Functionalized Tapered Multimode Fiber Coated With Dengue Virus NS1 Glycoprotein for Label Free Detection of Anti-Dengue Virus NS1 IgG Antibody

This proposed paper demonstrates the development of a bio-functionalized tapered multimode optical fiber for the detection of anti-Dengue virus NS1 Immunoglobin G (IgG). The tapered fiber with waist diameter and waist length of 10 $\mu \text{m}$ and 10 mm, respectively, promotes the penetration of light modes to the core-cladding interface, creating evanescent wave that are sensitive to changes on the fiber surface. As the fiber is bio-functionalized with Dengue virus (DENV) NS1 glycoprotein specific to anti-DENV NS1 IgG antibodies, alteration on the surface is selective towards the presence of IgG antibodies. The absorbance-based sensor obtained a sensitivity value of $7 \times 10$ −6 a.u/pg/mL with a detection time under 2 min. This paper shows great potential for real-world deployment which impact may bring Dengue diagnostics a step further.

Development and clinical evaluation of a highly accurate dengue NS1 rapid test: from the preparation of a soluble NS1 antigen to the construction of an RDT

Early diagnosis of dengue virus (DENV) is important. There are numerous products on the market claiming to detect DENV NS1, but these are not always reliable. In this study, a highly sensitive and accurate rapid diagnostic test (RDT) was developed using anti-dengue NS1 monoclonal antibodies. A recombinant NS1 protein was produced with high antigenicity and purity. Monoclonal antibodies were raised against this purified NS1 antigen. The RDT was constructed using a capturing (4A6A10, Kd=7.512±0.419×10−9) and a conjugating antibody (3E12E6, Kd=7.032±0.322×10−9). The diagnostic performance was evaluated with NS1-positive clinical samples collected from various dengue endemic countries and compared to SD BioLine Dengue NS1 Ag kit. The constructed RDT exhibited higher sensitivity (92.9%) with more obvious diagnostic performance than the commercial kit (83.3%). The specificity of constructed RDT was 100%. The constructed RDT could offer a reliable point-of-care testing tool for the early detection of dengue infections in remote areas and contribute to the control of dengue-related diseases.

Differentially Expressed Genes of Human Microvascular Endothelial Cells in Response to Anti-Dengue Virus NS1 Antibodies by Suppression Subtractive Hybridization

It has been previously shown that anti-dengue virus (DENV) nonstructural protein NS1 antibodies could act as autoantibodies that direct against one or more of the host's own proteins, which has potential implications for dengue hemorrhagic fever pathogenesis. In the present study, we have employed suppression subtractive hybridization (SSH) to identify the differentially expressed genes from human microvascular endothelial cells (HMEC-1) in response to anti-dengue virus type 2 NS1 antibodies (anti-DENV2 NS1 Abs). A total of 35 clones from the SSH cDNA library were randomly selected for further analysis using bioinformatics tools after vector screening. After searching for sequence homology in NCBI GenBank database with BLASTN and BLASTX programs, 23 obtained sequences with significant matches (E-values <1×10(-4)) in the SSH library. The predicted genes in the subtracted library include immune response molecules (CD59 antigen preproprotein preproprotein, MURR1), signal transduction molecules (Nuclear casein kinase and cyclin-dependent kinase substrate 1), calcium-binding proteins (S100A6, Annexin A2 isoform 1/2), and cell-membrane component (Yip1 domain family). From these clones, 5 upregulated genes were selected for differential expression profiling by real-time RT-PCR to confirm their upregulated status. The results confirmed their differential upregulation, and thus verified the success of SSHs and the likely involvement of these genes in dengue pathogenesis.

Production of anti-dengue NS1 monoclonal antibodies by DNA immunization

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 μg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.

Study of anti-dengue NS1 antibody by Western blot

The presence of anti-nonstructural protein (NS1) antibody in natural dengue infections has been suspected to be associated with development of dengue haemorrhagic fever (DHF). The Western blot technique was used to study the dynamics of the immune response to NS1 and to determine the frequency of anti-NS1 antibody among confirmed dengue patients in Indonesia, where DHF is common, and in Puerto Rico, where DHF is less frequently observed. Anti-NS1 antibody was rarely found in those with primary infections in either group. The antibody occurred at a significantly higher frequency in acute-phase serum samples from secondary infections in Indonesia than in those from Puerto Rico. No difference was observed, however, in Indonesian patients with secondary infection who had dengue fever or DHF.