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

Eduardo J M Nascimento,Allan Parker,Brooke Norwood,Ralph Braun,Eloi Kpamegan,Hansi J Dean

doi:10.3390/ijms222112004

Eduardo J M Nascimento, Allan Parker + Show 4 more

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https://doi.org/10.3390/ijms222112004

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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.

Highlights

Dengue virus (DENV) is a Flavivirus with four different but antigenically related serotypes (DENV1-4) that cause a wide range of symptoms from dengue fever, a debilitating flu-like disease, to the more severe and sometimes lethal manifestation of dengue hemorrhagic fever
The potency of neutralizing antibodies against DENV and other Flaviviruses (e.g., West Nile virus) is enhanced in the presence of proteins of the complement system (CS) [4,5,6], suggesting that the repertoire of antibody functions against DENV is more complex than the antibody–viral interactions measured in standard neutralizing antibody assays
We developed and characterized a novel, reliable, and easy to execute multiplex anti-DENV complement-fixing antibody assay based on the Luminex platform

Summary

Introduction

Dengue virus (DENV) is a Flavivirus with four different but antigenically related serotypes (DENV1-4) that cause a wide range of symptoms from dengue fever, a debilitating flu-like disease, to the more severe and sometimes lethal manifestation of dengue hemorrhagic fever. DENV affects tropical and subtropical areas of the world where the mosquito vector (mostly Aedes aegypti) is present. DENV exposure induces production of IgM, IgG, and IgA immunoglobulins that target pre-membrane (prM), envelope (Env), and other viral proteins, resulting in virus neutralization through inhibition of receptormediated entry and/or host membrane fusion [1,2,3]. The potency of neutralizing antibodies against DENV and other Flaviviruses (e.g., West Nile virus) is enhanced in the presence of proteins of the complement system (CS) [4,5,6], suggesting that the repertoire of antibody functions against DENV is more complex than the antibody–viral interactions measured in standard neutralizing antibody assays. There are three distinct pathways through which the CS can be activated, the classical pathway (CP) is initiated by antibody–antigen complexes or pattern recognition molecules (e.g., complement component 1q (C1q)) present in pathogen

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