Human antibody titers to Epstein-Barr Virus (EBV) gp350 correlate with neutralization of infectivity better than antibody titers to EBV gp42 using a rapid flow cytometry-based EBV neutralization assay

Abstract

Measurement of neutralizing antibodies to Epstein-Barr virus (EBV) is important for evaluation of candidate vaccines. The current neutralization assay is based on antibody inhibition of EBV transformation of B cells and requires 6 weeks to perform. We developed a rapid, quantitative flow cytometry assay and show that neutralizing antibody titers measured by the new assay strongly correlate with antibody titers in the standard transformation-based assay. Antibodies to EBV gp350 and gp42 have been shown to block infection of B cells by EBV. Using new assays to quantify antibodies to these glycoproteins, we show for the first time that human plasma contains high titers of antibody to gp42; these titers correlate with neutralization of EBV infectivity or transformation. Furthermore, we show that antibody titers to EBV gp350 correlate more strongly with neutralization than antibody titers to gp42. These assays should be useful in accessing antibody responses to candidate EBV vaccines.

Fig. 1

Neutralization of EBV B95-8/F infection by 72A1 monoclonal antibody (A) and human plasma (B). Relative infection (percentage) was determined by dividing the number of GFP-positive (infected) cells in the presence of antibody or plasma by the number of GFP-positive cells in the absence of antibody or plasma and converting to percentage for each antibody concentration (g/ml) or plasma dilution point (dilution). Each experiment was done in triplicate and non-linear regression analysis was performed. Symbols show the means at each point, lines show regression curves, and shaded columns represent the range of EDI₅₀ values in each experiment. Regardless of the amount of virus input, measured in green Raji units [GRU], the EDI₅₀ is constant.

Fig. 2

GFP-based infection neutralizing titration assay results for plasma samples from EBV seropositive (A) and EBV seronegative donors (B). Non-linear regression analysis was applied to each sample result. The dotted horizontal line in (A) is the percent of positive cells at 50% inhibition by the plasma, and the x intercept of the vertical dotted line is the dilution of antibody that inhibits infection by 50% (EDI₅₀). For this plasma the EDI₅₀ is 10^1.368 = 23.35. Samples were deemed negative (B) if the statistical software (Graphpad PRISM) failed to fit the data to a sigmoidal regression curve. Closed circles represent the average of triplicate values, and error bars show the range of percentage of positive cells at each dilution point.

Fig. 3

Comparison of GFP-based infection neutralization and conventional transformation neutralization assays. (A) Effective dilution of human plasma that inhibited infectivity by 50% (EDI₅₀) and transformation by 50% (EDT50) using the GFP infection-based and transformation-based neutralizing assay. Horizontal bars indicate means, vertical bars indicate 95% CI, closed circles or squares indicate EBV seropositive plasma, and open circles or squares indicate EBV seronegative plasma. (B) Correlation between GFP-based infection and conventional transformation neutralizing assay. Neutralization titers of 25 samples positive in both GFP-based infection and conventional transformation-based assay were plotted.

Fig. 4

Comparison of gp350 antibody titers with GFP-based infection neutralization, conventional transformation neutralization, and VCA antibody assays. (A) Anti-gp350 antibody titers by LIPS assay for EBV seropositive (closed circles) or seronegative (open circles) human plasma samples. Cut off value is shown as horizontal dotted line, which was determined as the mean + 2 SD of blank signal (closed squares). Correlation between gp350 antibody titer and GFP-based infection neutralization assay (B), conventional transformation-based neutralization assay (C), and EBV VCA IgG ELISA (D) for human plasma samples.

Fig. 5

Comparison of gp42 antibody titers with GFP-based infection neutralization, conventional transformation neutralization, and VCA antibody assays. (A) Anti-gp42 antibody titers by LIPS assay for EBV seropositive (closed circles) or seronegative (open circles) human plasma samples. Cut off value is shown as horizontal dotted line, which was determined as the mean + 2 SD of blank signal (closed squares). Correlation between gp42 antibody titers and GFP-based infection neutralization assay (B), conventional transformation-based neutralization assay (C), gp350 antibody assay, (D) and EBV VCA IgG ELISA (E) for human plasma samples.