Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) and Epstein-Barr virus (EBV) epitopes elicit a consistent humoral response in serum of multiple sclerosis patients, but the cross reactivity against the homologous myelin basic protein (MBP) and human interferon regulatory factor 5 (IRF5) has not been searched within the Cerebral Spinal Fluid (CSF). We evaluated in sera and CSF of patients with MS and with other neurological diseases (OND) the humoral response against EBV/MAP peptides and the IRF5/MBP. Our data showed that EBV and MAP peptides are able to induce a specific humoral immune response in MS patients compared to OND controls both in serum and in CSF. An intrathecal specific synthesis of IgG against MBP and their EBV and MAP homologous as indicated by the antibody index was observed in MS patients. The humoral response against EBV, MAP, MBP and IRF5 was significantly higher in MS patients compared to OND both in serum and in CSF. The higher presence of antibodies against MBP and their MAP and EBV homologous in CSF during relapses suggests a possible role of the pathogens in enhancing inflammation.

Figure 1. Antibody OD measured by indirect ELISA.

Forty-three patients, seventeen IND, eleven NIND and 5 UND were tested for their reactivity against plate-coated with EBNA1_400–413, MAP_0106c_121–132, MBP_85–98, in serum (A,C,E) and in CSF (B,D,F). The horizontal black bars represent the mean value, while p values are indicated by two headed arrows drown on the top of each distribution. Cut-off values for positivity, calculated by ROC analysis, are indicated by dashed lines.

Figure 2. Antibody OD measured by indirect ELISA.

Forty-three patients, seventeen IND, eleven NIND and 5 UND were tested for their reactivity against plate-coated with BOLF1_305–320, MAP_4027_18–32 and IRF5_424–434 in serum (A,C,E) and in CSF (B,D,F). The horizontal black bars represent the mean value, while p values are indicated by two headed arrows drown on the top of each distribution. Cut-off values for positivity, calculated by ROC analysis, are indicated by dashed lines.

Figure 3

Scatter plot showing the correlations between EBNA1_400–413 with MAP_0106c_121–132 in serum (A) in CSF (B). EBNA1_400–413 and MAP_0106c_121–132 with MBP_85–98 in serum (C,E) in CSF (D,F) in MS patients. Pearson’s correlation was calculated by Graph Pad 6.

Figure 4

Scatter plot showing the correlations between BOLF1_305–320 with MAP_4027_18–32 in serum (A) in CSF (B). BOLF1_305–320, MAP_4027_18–32 with IRF5_424–434 in serum (C,E) in CSF (D,F) in MS patients. Pearson’s correlation was calculated by Graph Pad 6.

Figure 5

Competition assay with MBP (A) and IRF5 (B) coated ELISA plates. (A) CSF from 2 MS patients and 1 IND were pre-incubated overnight with saturating concentrations [10 μM] of MAP2694_38–46 (negative control), EBNA1_400–413, MAP 106_121–132 and MBP 85–98 (positive control), The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide. (B) The same CSF were pre-incubated with MAP2694_38–46 (negative control), BOLF1_305–320, MAP_4027_18–32, and IRF5_424–434 (positive control). The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide.