Discrepancy in dengue virus neutralizing antibody titers between plaque reduction neutralizing tests with Fcgamma receptor (FcgammaR)-negative and FcgammaR-expressing BHK-21 cells

Abstract

Protective immunity against dengue virus (DENV) is best reflected by the presence of neutralizing antibodies. The conventional plaque reduction neutralizing test (PRNT) is performed using Fcgamma receptor (FcgammaR)-negative cells. Because FcgammaR plays a key role in antibody-dependent enhancement, we examined neutralizing antibody titers of mouse monoclonal antibodies and human serum samples in PRNTs using FcgammaRIIA-negative and FcgammaRIIA-expressing BHK cells. There was a discrepancy in dengue virus neutralizing antibody titers between PRNTs using FcgammaRIIA-negative versus FcgammaRIIA-expressing BHK cells. Neutralizing antibody titers to DENV-1 and DENV-2 tested with monoclonal antibodies, and with most of the human serum samples, were higher in assays using BHK cells than those using FcgammaRIIA-expressing BHK cells. The results suggest that neutralizing antibody titers determined using FcgammaRIIA-expressing cells may better reflect the protective capacity of anti-DENV antibodies, as the major target cells of DENV infection are FcgammaR-positive cells.

FIG. 1.

Plaque reduction neutralizing assays using BHK-21 cells and BHK-FcγRIIA cells. DENV-1 (A) and DENV-2 (B) were reacted with serially diluted mouse monoclonal 3H5 antibody in PRNT assays using BHK-21 and BHK-FcγRIIA cell lines in 12-well plates.

FIG. 2.

Patterns of plaque reduction against DENV in neutralization assays. (A) 4G2 MAb; (B) 3H5 MAb. ○, untransfected BHK-21 cells; ▴, BHK-FcγRIIA/2 cells; ▪, BHK-FcγRIIA/4 cells. Each curve is the mean of duplicate experiments.

FIG. 3.

Patterns of plaque reduction against DENV-1 in neutralization assays with human serum samples. DENV-1 was reacted with human serum samples 2-fold serially diluted from 1:5 to 1:2,560. Graphs are presented according to serum sample number. The characterization of samples is delineated in Tables 2 and 3. ○, untransfected BHK-21 cells; ▴, BHK-FcγRIIA/2 cells; ▪, BHK-FcγRIIA/4 cells. Each curve is the mean of duplicate experiments.

FIG. 4.

Patterns of plaque reduction against DENV-2 in neutralization assays with human serum samples. DENV-2 was reacted with human serum samples 2-fold serially diluted from 1:5 to 1:2,560. Graphs are presented according to serum sample number. The characterization of samples is delineated in Tables 2 and 3. ○, untransfected BHK-21 cells; ▴, BHK-FcγRIIA/2 cells; ▪, BHK-FcγRIIA/4 cells. Each curve is the mean of duplicate determinants.