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

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.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in demyelination and neurodegeneration 1 . The disease develops in genetically predisposed individuals in response to environmental factors, most likely viral and bacterial infections 1 . However, recent studies advocate the possible combined roles of Mycobacterium avium subsp. paratuberculosis (MAP) and Epstein Barr virus (EBV) in the process of autoimmunity inducing MS pathology 2,3 . In fact, it was demonstrated that peptides deriving from these pathogens could be cross-recognized by antibodies (Abs) targeting self-epitopes 2,3 . In particular it was detected a strong humoral response against peptides from the latent protein of Epstein Barr Virus (EBNA1 400-413) , the homologous mycobacterial MAP_0106c 121-132 and the human Myelin Basic Protein (MBP [85][86][87][88][89][90][91][92][93][94][95][96][97][98] in MS patients compared to healthy controls (HCs). Also, an interesting common humoral response was reported against a relevant EBV epitope from EBV lytic protein (BOLF1 305-320 ) and two homologous peptides belonging to MAP_4027 protein (MAP_4027 [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] ) and Human Interferon Regulatory Factor 5, IRF5 protein (IRF5 424-434 ) 3 . A cross-recognition of the mentioned peptides was also demonstrated. In this work we wanted to explore if we could find the same humoral response in CSF and serum against EBV epitopes deriving from EBV lytic and latent proteins, MAP and human homologous proteins in MS patients and in other neurological disease controls. The humoral response against EBV and MAP in CSF could contribute to understand an immunological dysregulatation in the CSF of MS patients. The presence of intrathecal IgGs, is still considered an evidence for the involvement of infectious agents in MS pathogenesis, although their specificity is largely unknown 4 . This study was carried out on samples from patients with MS, inflammatory neurological disease (IND) or non inflammatory neurological disease (NIND) and patients where a diagnosis was not reached, indicated as undetermined neurological disease (UND),aiming to: a) measure circulating serum and CSF Abs against EBV and MAP peptides and their human homologous; b) quantify and correlate the serum IgG levels to the CSF IgG production; c) investigate the IgG cross reaction against the epitopes investigated. Molecular mimicry between immunodominant epitopes deriving from bacterial and viral persistent antigens may be a decisive factor in directing autoimmunity to self-antigens in MS patients. For this reason it was important to explore if the epitopes from EBV and the other homologous MAP antigens were able to induce a humoral reactivity both in CSF and sera. The results could contribute to the understanding of chronic brain inflammation that contribute to MS pathogenesis.

CFS/Serum Albumin ratio and Link index.
For all samples, Link index as a generic marker of intrathecal IgG synthesis, CSF/serum albumin ratio (Q Alb) as a marker of BBB integrity and percentage of samples with different type of BBB damage were evaluated and shown in Table 1. No statistically significant damage was observed in the BBB of the MS group compared to the other groups as evidenced by the Link index and the CFS/ albumin ratio.  (Fig. 2F). We considered also a double Abs positivity Serum/CSF in MS samples compared to that found in IND/NIND against the single peptides and their combination ( Table 2). A double positivity for EBNA1 and BOLF1 peptides was found among the 33% and 37%, respectively in serum and CSF whereas only 9% of IND/NIND/UND controls was positive (p = 0.025 and 0.007 respectively) ( Table 2). Same pattern was observed for the double positivity to MAP_106c 121-132 and MAP_4027 [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] in serum and CSF of MS (37% and 30%, respectively) against the 15% in serum and 4% in CSF of IND/NIND/UND subjects (p = 0.04 and 0.0024 respectively) ( Table 2). A strong positive association was found for triple positivity (21%) both in serum and in CSF to BOLF1 305-320 , MAP_4027 18-32 and IRF5 424-434 in MS patients in comparison to that (0%) of IND/NIND/ UND subjects (p = 0.0042) ( Table 2). Moreover, CSF positivity to EBNA1 400-413 , MAP_106c 121-132 and MBP 85-98 was associated to MS patients with relapse compared to MS patients without relapse (Table 3).

Correlation analyses.
A correlation analyses was performed to establish association between the peptide positivity. We found a good correlation for EBNA1 400-413 peptide with the homologous MAP_106c 121-132 peptide in serum (R 2 = 0.57; P < 0.0001) and a little less (R 2 = 0.35; p < 0.0001) in CSF (Fig. 3A,B). Interestingly, the correlation analysis showed a stronger correlation between MBP 85-98 and EBNA1 400-413 in CSF (R 2 = 0.56; p < 0.0001)  Table 1. Link index as a generic marker of intrathecal IgG synthesis, CSF/serum albumin ratio (Q Alb) as a marker of BBB integrity and percentage of samples with different type of BBB damage are shown.
Competition assay. To demonstrate the cross-reactivity between MAP/EBV and self epitopes, we developed two competition assays: one coating the MBP 85-98 on plate and the other coating the IRF5 424-434 on plate (Fig. 5A,B, respectively). Concerning MBP 85-98 -coated competition assay (Fig. 5A), in all the CSF subjects (MS#1,  (Fig. 5A,B). Taken together, these results demonstrate, that Abs anti-EBNA1 and anti-MAP106 target the same conformational MBP epitope and also demonstrate that Abs anti-BOLF1 and anti-MAP 4027 targeting the same conformational IRF5 epitope are cross-reactive in CSF.

Peptide IgG-specific antibody index (AI) in MS patients. Antibody Index (AI) for all MS patients was
calculated with the following formula AI = QIgG[spec]/Qlim 7 . A high number of positive samples against the selected peptides in the MS group had an AI > 1.5 that demonstrate an intratecal IgG-specific antibody production against EBNA1 400-413 , MAP 106, MBP 85-98 and BOLF1 305-320 , MAP_4027 18-32 , IRF5 424-434 peptides, as shown in Table 4.

Discussion
MS aetiology remains unclear, and the current accepted theory for its pathogenesis assigns the main role to the destruction of the myelin-proteolipid shell of axons resulting in autoimmune reactions supposedly induced by a different viral or bacterial infection 1,6,8 . Although activation of the T-cell response has a crucial role in MS pathogenesis, the B-cell response is equally responsible for the development of the disease by the enhanced synthesis of immunoglobulins usually IgGs 9 . It has been shown that the CSF from MS patients contains oligoclonal immunoglobulins (IgG), which are synthesized within the central nervous system and presumably related to the immune dysfunction, a characteristic feature of MS. The aim of this study was to investigate the presence of a specific humoral response mounted against peptides derived from EBV and MAP antigens homologues to host proteins in serum and in CSF of MS compared to Inflammatory and No inflammatory neurological disease. We investigated different aspects of the association of CSF and serum EBV and MAP positivity in MS and controls. Our studies reported the ability of specific peptides from EBNA1, BOLF1, MAP and their human homologues to induce a strong humoral response in MS patients in comparison to healthy controls, and the existence of a cross-recognition between Abs recognizing these homologues peptides [10][11][12] . Also these works showed up that EBV and MAP are capable of inducing the production of autoantibodies targeting different MS correlated epitopes 13 . In this study we look for the presence of intrathecal immune response against EBV, MAP and the homologous human peptides. To check the BBB integrity, the Antibody Index (AI) as the marker of a specific intrathecal IgG synthesis against the selected peptides and CSF/serum albumin ratio (Q Alb) were evaluated. Results showed an high number of MS patients with intrathecal IgG synthesis against EBV and MAP with a AI > 1.5, this associated with a better BBB integrity in MS patients in comparison to that of IND, NIND and UND controls (Table 1)    that IgGs against peptides from EBV, MAP and human homologues peptides such as MBP 85-98 and IRF5 424-434 were higher in MS patients than in IND/NIND/UND controls in both CSF and serum. Interestingly, a higher correlation was found between the levels of IgGs against EBV, MAP and their homologous peptides in CSF rather than in serum 5,10,12 . Therefore, we set up a competition assay between the human peptides and their homologous from EBV and MAP in CSF. Our results highlighted that autoantibodies recognizing MBP and IRF5 were able to cross-react with the homologous peptides from EBV (EBNA1, BOLF1) and from MAP (MAP106, MAP4027), possibly by a molecular mimicry mechanism. The soundness of the results was confirmed by the correlation results, the fact that a triple positivity was found, both in serum and in CSF, against BOLF1 305-320 , MAP_4027 18-32 and IRF5 424-434 in MS patients in comparison to the absence in IND/NIND/UND subjects confirmed our hypothesis that MAP and EBV could ignite the Sundström et al. also considered as a risk factor the interaction of antibodies against EBNA-1 specific domains and HLA DRB1*1501 14 . In our hands we didn't find any correlation between a specific HLA and the immunoreactivity with our peptides (data not shown). Recently, another group showed that a productive EBV infection in the peripheral blood could facilitate entry of viral particles and/or newly infected B cells into the CNS 15 . This finding may support our result on the specific ABs against EBV peptides in CSF of MS patients. Our findings emphasize the significance of CSF and serum levels of anti-EBV and MAP IgG. Furthermore, the cross-reaction of MBP 85-98 supports the possible role of IgG against myelin basic protein in MS disease. Therefore, it is not surprising that several studies report a statistically significant correlation of Abs titers of MBP in MS patients rather than in other neurological disease controls 5 . This data suggest that EBV and MAP may be capable of inducing the production of autoantibodies targeting different MS correlated epitopes expressed in CNS. In conclusion the detection of anti-EBV and anti-MAP IgG in MS patients provided additional evidence of the possible synergistic role of these microrganisms to induce the pathology. Thus, the detection of Abs against EBV and MAP in CSF of MS patients can be considered as an additional criterion (immunological parameter) crucial to better understand MS pathology.

Materials and Methods
Subjects. The study protocol was approved by the ethic committee of the University of Cagliari, Italy. All the methods were carried out in "accordance" with the approved guidelines. All the participants provided written consent. CSF and serum samples prospectively collected from MS and Other Neurological Disease composed (OND) by Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND) patients were obtained for diagnosis purposes and measured under equal conditions. CSF samples were obtained by a traumatic lumbar puncture performed for diagnosis purposes in the absence of contraindications. All MS samples derived from 43 patients (24 females and 19 males; mean age ± SD was 39 ± 14.0 years) that fulfilled the revised McDonald diagnostic criteria 16 . At the time of the study, 39 patients were diagnosed as relapsing remitting MS, 4 as secondary progressive MS. The duration of the disease was 6 ± 5.4 years, the average age for MS onset was 29.0 ± 10.5 and the expanded disability status scale (EDSS) values ranged from 0 to 7.0 with a 1.9 average. Other neurological diseases (OND) serum and CSF samples were collected from 33 patients (14 females and 19 males) so divided: 17 IND (5 females and 12 males, mean age ± SD was 39 ± 23.1 years ), 11 NIND (6 females and 5 males, mean age ± SD was 41 ± 27.1 years) and 5 UND (2 females and 3 males, mean age ± SD was 71 ± 11 years).
ELISA. Indirect Enzyme-Linked Immunosorbent Assays (ELISA) was carried out to detect specific Abs for all the synthetic peptides (assayed at 10 μg/ml) included in the study as previously reported 17,18 indirect