Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays

Abstract

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

Rapid Neutralization Testing System for Zika Virus Based on an Enzyme-Linked Immunospot Assay

“…33 Therefore, a rapid and high-throughput neutralization test for ZIKV is urgently needed, which is significant for promoting the development of vaccines. However, currently, the Nt-CPE assay, the classical neutralization method for flaviviruses, 14,15 is time-consuming (with 5−7 days) and laborintensive and has low-throughput, so it can hardly meet the demands of the detection of large numbers of specimens in vaccine development or serosurvey. Researchers in this field have tried to overcome this limitation.…”

“…In 2017, a rapid ZIKV diagnostic assay (with a turnaround time within 48 h) was reported by Shan et al 16 However, the luciferase viruses utilized to quantify the neutralizing antibody in their method could not represent the clinical virus strain completely. The enzyme-linked immunospot assay (ELISPOT) has been widely used and demonstrated to be a highly sensitive, objective, and high-throughput immunological method for detecting neutralizing antibodies against many kinds of viruses, including DENV, which is also a flavivirus; 14,17,18 enterovirus 71 (EV71); 19 coxsackievirus A16 (CVA16); 20 coxsackievirus A10 (CVA10); 21 coxsackievirus B3 (CVB3); 22 rotavirus; 23 HSV-1. 24 However, whether this assay can be applied to ZIKV remains unknown.…”

How Can Elispot Add Information to Improve Knowledge on Tropical Diseases?

“…It can also be used as a microneutralization plaque assay as described by several authors [ 66 , 67 , 68 , 69 , 70 , 71 ]. Liu et al (2001) compared a plaque reduction neutralization assay (PRNT) with a microneutralization enzyme-linked immunospot-based assay (Elispot-MNT) to measure the neutralizing activities of antibodies specific to dengue virus envelope protein and they pointed some advantages for Elispot-MNT: 1- Elispot-MNT is faster than PRNT, has a higher throughput, and is more objective [ 69 ].…”

Functional properties of DENV EDIII-reactive antibodies in human DENV-1-infected sera and rabbit antiserum to EDIII

“…In vitro enhancement assay by NS1 capture ELISA-based ADE assay (ELISA-ADE). The assessment of ADE by measuring DENV NS1 production in Fcγ receptor-expressing K562 cell culture supernatants was performed by modified ELISA-ADE as previously described (29). Briefly, serially diluted serum samples were incubated with an equal volume of DENV at a multiplicity of infection of 0.5 or 0.125 in 96-well plates for 1 h at 37˚C.…”