Development and Characterization of a Multiplex Assay to Quantify Complement-Fixing Antibodies against Dengue Virus

Abstract

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as “complement-fixing antibodies” and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

“…The Luminex assay was conducted by FlexMap 3D (Luminex, Austin, TX, USA), and the conjugation of VLP was previously reported ( 70 ). Briefly, 65 μg ZIKV and DENV-VLP was conjugated to 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, ECD/N-hydroxy-sulfo-succinimide, NHS (Thermo Fisher, Waltham, MA, USA), and activated in 12.5 million MagPlex beads (Luminex, Austin, TX, USA) in 50 mM 2-(N-morpholino)ethanesulfonic acid buffer, pH 7.0 or 6.0, for 120 min at room temperature.…”

Antibodies Produced in Response to a Live-Attenuated Dengue Vaccine Are Functional in Activating the Complement System

“…CFAs were measured as described elsewhere [ 38 ]. DENV-binding IgG1, IgG3, IgM, and IgA Luminex assays (Luminex Corporation) were developed and characterized ( Supplementary Tables 1–6 and Supplementary Figures 1–4 ) using the same DENV virus-like particle (VLP)-coupled magnetic microspheres, plates, and assay buffers used in the anti-dengue CFA assay [ 38 ]. Sample heat inactivation, incubation with VLP-coupled microspheres, and shaking speed were similar to those used with the CFA assay [ 38 ].…”

“…DENV-binding IgG1, IgG3, IgM, and IgA Luminex assays (Luminex Corporation) were developed and characterized ( Supplementary Tables 1–6 and Supplementary Figures 1–4 ) using the same DENV virus-like particle (VLP)-coupled magnetic microspheres, plates, and assay buffers used in the anti-dengue CFA assay [ 38 ]. Sample heat inactivation, incubation with VLP-coupled microspheres, and shaking speed were similar to those used with the CFA assay [ 38 ]. Sample serial dilution range and secondary antibody concentrations are shown in Supplementary Table 7 .…”

“…See Supplementary Material for additional details. Limit of detection, lower limit of quantitation (LLOQ), and precision for the CFA effector function and virus-binding Luminex assays (IgG1, IgG3, IgM, and IgA) are described in Nascimento et al [ 38 ] and Supplementary Tables 1–6 , respectively.…”

“…We investigated the ability of TAK-003 to elicit production of complement-fixing antibodies (CFAs) based on C1q fixation against all 4 DENV serotypes [ 38 ], and determined the relationship with anti-DENV antibody subclasses (IgG1 and IgG3) and isotypes (IgA and IgM). Additionally, we investigated the relationship between CFA responses with nAb titers, virus-binding total IgG concentration, and IgG avidity.…”