Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the β-ladder domain.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the β-ladder domain (amino acid residues 178-273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1

Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident

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