Epstein-Barr virus neutralizing and early antigen antibodies in multiple sclerosis

Abstract

Background: Our objective was to determine whether antibodies against the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1), early antigen (EA), and EBV neutralizing antibodies (NeutAb) are altered in multiple sclerosis (MS).

Methods: We measured EBNA-1 IgG, EA IgG, and EA IgA using quantitative ELISA. We measured NeutAb using a quantitative competitive ELISA. We studied 80 patients with MS, 80 matched controls, and 19 patients with MS with samples collected both whilst stable and in relapse.

Results: Epstein-Barr virus nuclear antigen-1 IgG and EA IgA were increased in MS compared to controls. The EBNA-1 index value was 23.3 ± 18.3 in the patients with MS (mean ± SD) and 16.3 ± 17.4 in the controls (P = 0.007, paired t-test). EA IgA had a median value of 1.964 in the patients with MS and 1.248 in the controls (P = 0.029, Wilcoxon signed rank test). EA IgG and NeutAb were not significantly different. None of the antibody levels were altered in relapse. The correlation between concentrations of different antibodies was minimal.

Conclusions: IgG antibodies to EBNA-1 are significantly increased in MS. IgA antibodies against EBV EA are also increased. The EBV neutralizing antibody response is similar in MS and controls.

Figure 1. A. Immunoprecipitation of gp350 with 72A1 antibody

The first lane is 5 μl of the crude membrane fraction, the second lane is the immunoprecipitate of the membrane fraction with 5 μg 72A1, and the third lane is 72A1 antibody alone. The crude membrane fraction has a protein at >250 kilodaltons which is immunoprecipitated with 72A1. Numbers indicate positions of the molecular weight markers. Gradient gel 4-12% polyacrylamide stained with Coomassie and Alcian. B. Example of neutralizing antibody competitive ELISA. See text for details. C. Reproducibility of the NeutAb ELISA. Results of repeat measurements of the same serum sample at different times. Both axes are on a logarithmic scale.

Figure 2. Antibody responses in MS and controls

The box encloses the values for the 25^th to 75^th percentiles, the whiskers indicate the 10^th and 90^th percentiles, and the dots indicate the 5^th and 95^th percentiles. The median is indicated by the line across the box. A. EBNA-1 IgG, values are given as index values relative to a calibrator sample, the difference between groups is significant, p=0.007, paired t test. B. EA IgG, values are given as index values relative to a calibrator sample. C. EA IgA, values given are the sample OD divided by the OD of a single comparison sample used for all experiments. The difference between groups is significant, p=0.029, Wilcoxon signed rank test. D. NeutAb IgG, values measured in microgram equivalents of the monoclonal neutralizing antibody 72A1, y-axis is logarithmic and values below the limit of the assay are plotted at 0.01.

Figure 3. Changes in antibody titers with relapse

Each point represents a single MS patient, the value while stable is on the x-axis and the value during relapse is on the y-axis. Subjects with higher values in relapse will be plotted to the upper left of the diagonal line, lower values in relapse will be below and to the right of the diagonal. The units are the same as in Figure 2.