Revisiting IgG Antibody Reactivity to Epstein-Barr Virus in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Its Potential Application to Disease Diagnosis

Abstract

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.

Keywords: Epstein-Barr virus; Myalgic Encephalomyelitis/Chronic Fatigue Syndrome; antigen mimicry; biomarker discovery; patient stratification.

Figure 1

Preliminary multivariate analysis of the data. Scatterplots of the first two principal components (left plots) and the ROC curve and its AUC of the respective LDA (right plots) when comparing all the ME/CFS patients to healthy controls (A), ME/CFS patients with an infectious trigger to healthy controls (B), ME/CFS patients with a non-infectious or unknown trigger to healthy controls (C), and ME/CFS patients with an infectious trigger to the remaining patients (D). The percentage of the variance explained by each principal component is shown in each axis within brackets.

Figure 2

Antibody-wide association analyses when comparing all the ME/CFS patients to healthy controls (A), ME/CFS patients with an infectious trigger to healthy controls (B), ME/CFS patients with a noninfectious or unknown trigger to healthy controls (C), and ME/CFS patients with an infectious trigger to the remaining patients (D). The x-axes comprise each antibody while the y-axes represent the −log₁₀(adjusted p-value) of the respective association. In the x-axes, the antibodies were ordered alphabetically first by the protein name and then by the starting point of the antigen within the protein. Adjusted p-values were calculated according to the Benjamini-Yekutieli procedure for a global FDR of 5% under the assumption of dependent data. Dashed line represents the threshold for statistical significance (i.e., −log₁₀(FDR = 0.05)) and −log₁₀(adjusted p-values) > 1.30 were considered statistically significant.

Figure 3

Statistical analysis of the antibody levels related to EBNA4_0529, EBNA6_0066, and EBNA6_0070. (A) Boxplots of the data per study group. (B) Scatterplots and the respectively Spearman's correlation coefficients (R) in the whole dataset. (C) Amino acid sequences of EBNA4_0529, EBNA6_0066, and EBNA6_0070.

Figure 4

Analysis of the final classification model for predicting ME/CFS patients with an infectious trigger when compared to healthy controls. (A) Contour plots of the probability of being a patient as a function of age and EBNA6_0070 antibody levels, for men and women, respectively. The prediction values were calculated by fixing log₁₀(EBNA4_0529) at the respective mean value. (B) ROC curves and the respective AUC (95% confidence interval shown within brackets) when using the model to compare different groups of ME/CFS patients to healthy controls.