Dengue and the lectin pathway of the complement system

Dengue is a mosquito-borne viral disease causing significant health and economic burdens globally. The dengue virus (DENV) comprises four serotypes (DENV1-4). Usually, the primary infection is asymptomatic or causes mild dengue fever (DF), while secondary infections with a different serotype increase the risk of severe dengue disease (dengue hemorrhagic fever, DHF). Complement system activation induces inflammation and tissue injury, contributing to disease pathogenesis. However, in asymptomatic or primary infections, protective immunity largely results from the complement system\u27s lectin pathway (LP), which is activated through foreign glycan recognition. Differences in N-glycans displayed on the DENV envelope membrane influence the lectin pattern recognition receptor (PRR) binding efficiency. The important PRR, mannan binding lectin (MBL), mediates DENV neutralization through (1) a complement activation-independent mechanism via direct MBL glycan recognition, thereby inhibiting DENV attachment to host target cells, or (2) a complement activation-dependent mechanism following the attachment of complement opsonins C3b and C4b to virion surfaces. The serum concentrations of lectin PRRs and their polymorphisms influence these LP activities. Conversely, to escape the LP attack and enhance the infectivity, DENV utilizes the secreted form of nonstructural protein 1 (sNS1) to counteract the MBL effects, thereby increasing viral survival and dissemination

Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection

Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection

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

Performance of Two Commercial Dengue NS1 Rapid Tests for the Diagnosis of Adult Patients with Dengue Infection

Objective: To determine the diagnostic performance of two commercially available dengue NS1 antigen rapid detection tests (RDTs); namely the SD BIOLINE Dengue NS1 RDT and the ImmuneMed Dengue NS1 Rapid, for the diagnosis of adult patients with dengue infection. Methods: The study was performed by using archived samples from 237 patients with the laboratory-confirmed dengue infection. Archived, well-characterized samples from an additional 208 febrile individuals from Thailand were used as the control group. Reference testing to confirm the diagnosis in dengue patients included RT-PCR and in-house NS1 ELISA, in-house IgM and IgG capture ELISAs. Results: The sensitivity of the SD BIOLINE Dengue NS1 RDT was 100%, and the specificity was 99% (95%CI 96.6 to 99.7%). False positive was found in 2 samples from patient with scrub typhus. The sensitivity and specificity of the ImmuneMed Dengue NS1 Ag Rapid were 97.4% (95% CI, 95.5 to 99.5%) and 96.6% (95%CI 93.5 to 98.4%) respectively. False positive results were found in 7 patients with murine typhus, scrub typhus, and influenza.   Conclusion: Both RDTs showed comparable sensitivity and specificity in this study population. Data from this study could be used to facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks

Binding of Flavivirus Nonstructural Protein NS1 to C4b Binding Protein Modulates Complement Activation

The complement system plays a pivotal protective role in the innate immune response to many pathogens including flaviviruses. Flavivirus nonstructural protein 1 (NS1) is a secreted nonstructural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but absent from viral particles. Previous work has defined an immune evasion role of flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. In this study, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile, or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of flavivirus infection. The Journal of Immunology, 2011, 187: 424-433

Analytical and diagnostic performance characteristics of reverse-transcriptase loop-mediated isothermal amplification assays for dengue virus serotypes 1-4: A scoping review to inform potential use in portable molecular diagnostic devices.

Dengue is a mosquito-borne disease caused by dengue virus (DENV) serotypes 1-4 which affects 100-400 million adults and children each year. Reverse-transcriptase (RT) quantitative polymerase chain reaction (qPCR) assays are the current gold-standard in diagnosis and serotyping of infections, but their use in low-middle income countries (LMICs) has been limited by laboratory infrastructure requirements. Loop-mediated isothermal amplification (LAMP) assays do not require thermocycling equipment and therefore could potentially be deployed outside laboratories and/or miniaturised. This scoping literature review aimed to describe the analytical and diagnostic performance characteristics of previously developed serotype-specific dengue RT-LAMP assays and evaluate potential for use in portable molecular diagnostic devices. A literature search in Medline was conducted. Studies were included if they were listed before 4th May 2022 (no prior time limit set) and described the development of any serotype-specific DENV RT-LAMP assay ('original assays') or described the further evaluation, adaption or implementation of these assays. Technical features, analytical and diagnostic performance characteristics were collected for each assay. Eight original assays were identified. These were heterogenous in design and reporting. Assays' lower limit of detection (LLOD) and linear range of quantification were comparable to RT-qPCR (with lowest reported values 2.2x101 and 1.98x102 copies/ml, respectively, for studies which quantified target RNA copies) and analytical specificity was high. When evaluated, diagnostic performance was also high, though reference diagnostic criteria varied widely, prohibiting comparison between assays. Fourteen studies using previously described assays were identified, including those where reagents were lyophilised or 'printed' into microfluidic channels and where several novel detection methods were used. Serotype-specific DENV RT-LAMP assays are high-performing and have potential to be used in portable molecular diagnostic devices if they can be integrated with sample extraction and detection methods. Standardised reporting of assay validation and diagnostic accuracy studies would be beneficial

Ultrastructural Features of Human Liver Specimens from Patients Who Died of Dengue Hemorrhagic Fever

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA–indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed

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