Development of an Improved Epstein-Barr Virus (EBV) Neutralizing Antibody Assay to Facilitate Development of a Prophylactic gp350-Subunit EBV Vaccine

Abstract

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed an improved EBV-GFP based neutralization assay for such a vaccine's pre-clinical and clinical validation to measure EBV specific neutralizing antibodies in human donors. The supplementation of guinea pig complement of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay allowed the detection of complement-dependent neutralizing antibodies using a panel of heat-inactivated healthy human sera. Anti-gp350 antibody titers, which were evaluated using a previously optimized anti-gp350 IgG ELISA assay, were moderately correlated to the FFA-based neutralization titers. Overall, this improved high-throughput neutralization assay is capable of characterizing the serologic neutralizing antibody response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

Keywords: Anti-gp350 IgG ELISA; Epstein-Barr Virus; Gp350; Neutralization assay; Vaccine.

Figure 1

Quantitation of EBV neutralization titers in human sera. A. Comparison of neutralization titer tested by Fluorescent Focus Assay (FFA)-based or Flow cytometry (FACS)-based neutralization assay with or without guinea pig complement. Healthy human donor sera samples obtained from AllCells, LLC and Bioreclamation, LLC (n=39) were tested in two different assay formats: FFA-based neutralization in SVK-CR2, the CR2-expressing transfected human epithelial cell line, or FACS-based neutralization assay in Raji, the human B lymphoblastoid cell line. In each assay format, the human sera were also tested under two conditions, supplemented with or without 1% guinea pig complement. The limit of the detection of both assay formats is 3.32 Log2 neutralization titer. Two folds of the limit of the detection of both assay formats are 4.32 Log2 neutralization titer. B. Correlation of EBV anti-gp350 IgG ELISA and the functional FFA-based neutralization assay supplemented with 1% guinea pig complement. Assay correlation was established with 32 human sera which tested positive in both assays (Pearson r value=0.5844, P value =0.0004).