An Automated Dengue Virus Microneutralization Plaque Assay Performed in Human Fcγ Receptor-expressing CV-1 Cells

Abstract

We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI-SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcgammaRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection.

Discrepancy in Dengue Virus Neutralizing Antibody Titers between Plaque Reduction Neutralizing Tests with Fcγ Receptor (FcγR)-Negative and FcγR-Expressing BHK-21 Cells

“…In the presence of Fc␥R, the infection enhancement effect may hamper neutralization (9). DENV immune complexes that formed with heterologous antibodies were less susceptible to neutralization in the presence of Fc␥RIIA-expressing BHK-21 cells, which is consistent with earlier findings by other investigators who used Fc␥RIIA-expressing CV-1 cells (13,16). In contrast, DENV-1 immune complexes formed with DENV-1 antibodies, or DENV-2 immune complexes formed with DENV-2 antibodies (homologous DENV immune complexes) were susceptible to neutralization in both BHK-Fc␥RIIA cells and parent BHK-21 cells.…”

“…The PRNTs using BHKFc␥RIIA cells satisfy a criterion for an acceptable alternative to conventional neutralization assays: the assay detects the sum of neutralizing and enhancing activities as neutralizing titers in the presence of Fc␥RIIA. At the same time, the simplicity and ease of performance using the cell lines in the present study meet or exceed those of previous studies (8,13,14,16). The results suggest that PRNTs using BHK-Fc␥RIIA cells could be a feasible alternative for the detection of neutralizing titers of DENV.…”

Comparison of Plaque- and Enzyme-Linked Immunospot-Based Assays To Measure the Neutralizing Activities of Monoclonal Antibodies Specific to Domain III of Dengue Virus Envelope Protein

“…Nevertheless, the conventional PRNT remains time-consuming and labor-intensive, with low throughput. To overcome these deficiencies of PRNT, newer tests assessing dengue virus neutralization are being developed, such as a microfocus reduction neutralization test (mFRNT) (8), microneutralization assays based on an ELISA (17), flow cytometry (10), and ELISPOT (15). However, any new approach to titration of dengue virus neutralizing antibodies will need to be validated against the standard PRNT.…”

“…The enzyme-linked immunospot based microneutralization test (ELISPOT-MNT), first described by Shanaka et al (15), was performed with minor modifications according to our optimal experiments (Fig. 1).…”

“…Recently, Shanaka et al (15) developed an enzyme-linked immunospot-based microneutralization assay (ELISPOT-MNT) to detect the viral antigen in infected cells and a 96-well enzymelinked immunospot readout instrument to measure the spots produced in an indirect immunostaining method. The extent of infection can be observed easily in ELISPOT-MNT by counting the spots; this is comparable to counting the plaques developed in the classic PRNT, but the former test offers an automated and highthroughput way for measuring neutralizing antibodies, which is also more objective.…”