We showed that most active and chronic-active MS plaques replete with perivascular B-lymphocyte cuffs and single MS CSF B-lymphocytes and plasma cells do not contain EBV-specific transcripts. EBV-specific transcripts examined were as follows: EBER-1, the most abundant transcript in latently infected cells19
; latent transcripts EBNA-2 and LMP-1; and lytic state transcript BFRF-1.20
Unlike other human herpesviruses, EBV is associated with multiple types of latency. Type 0 latency, in which only the EBER-1 transcript is found, is seen in circulating nondividing B-lymphocytes of healthy seropositive individuals, while types I, IIa, IIb, and III, in which EBER-1 and other EBV transcripts are found, is seen in Burkitt and Hodgkin lymphomas, chronic lymphocytic B-cell leukemia, and posttransplant lymphoproliferative disease.21,22
EBER-1 transcript is present in all forms of EBV latency.
Real-time PCR did not detect EBER-1 transcript in any single B-lymphocyte or plasma cell examined in MS CSF, and an extensive analysis of 15 acute, active, and chronic-active MS plaques revealed no EBER-1 transcripts. Additional EBV gene expression analysis of 3 plaques known to be EBV DNA-positive identified EBER-1 as the only EBV-specific transcript present. This transcription profile is characteristic of type 0 EBV latency, indicating that B-lymphocytes in the few plaques containing EBV are of the same latency type as that found in circulating nondividing B-lymphocytes of healthy seropositive individuals.
ELISA and immunostaining detected anti-EBV antibodies in CSF of MS and control patients with non-MS inflammatory CNS diseases. The anti-EBV AI, an indicator of intrathecal anti-EBV antibody production at AI values greater than 1.5,12
identified intrathecal synthesis in a few patients with MS and controls, but there was no significant difference between the patient groups in the frequency of anti-EBV IgG intrathecal synthesis. Anti-EBV oligoclonal IgG, reflecting substantial intrathecal anti-EBV antibody synthesis, is rare in MS.23,6,7
For example, about 13% of 132 patients with MS, and none of 125 control patients with noninflammatory neurologic disease, had oligoclonal IgG in their CSF to anti-EBNA-1 and anti-BRRF2 peptides.6
In another study, the CSF in 7 of 16 patients with MS contained EBV-specific oligoclonal IgG, although in 6 of these 7 patients with MS, anti-EBV bands were few and faint.7
It remains to be determined whether a subset of patients with other CNS inflammatory diseases whose CSF contains oligoclonal IgG also contains any EBV-specific oligoclonal IgG. Meanwhile, the rare intrathecal humoral immune response against EBV in MS CSF indicates that the virus is unlikely to be linked with disease pathogenesis. Besides anti-EBV antibodies, antibodies to measles, rubella, varicella zoster virus, and herpes simplex virus are also synthesized intrathecally in some patients with MS and patients with CNS infections,24,25
a finding that has been explained by infiltration of antibody-secreting B cells into the CNS of patients with infectious and inflammatory CNS diseases. Patients with high levels of intrathecal IgG production are more likely to have any one of the antiviral antibodies in their CSF25
than patients with low intrathecal IgG synthesis. Polyclonal activation of long-lived memory B cells has been observed after secondary immunization26,27
and could explain the infrequent detection of intrathecal antiviral antibodies, particularly in chronic inflammatory CNS disease. Thus, the presence of antibodies to diverse viruses in MS CSF and patients with other infectious and inflammatory diseases of the CNS represents an immune reaction common to infectious and inflammatory CNS diseases.
Most importantly, rAbs generated from clonally expanded plasma cells in MS CSF did not bind to EBV. CSF plasma cells, absent in healthy human CSF, are unique to the CSF of patients with infectious or inflammatory CNS diseases, and the antibodies they produce are directed against specific antigens involved in the pathogenesis of disease. Clonally expanded MS CSF plasma cells are generated through an antigen-driven germinal center-like maturation and differentiation process,8
and are the source of intrathecally synthesized oligoclonal bands found in MS CSF.28
The specificity of MS CSF rAbs likely duplicates the specificity of MS CSF oligoclonal bands that are thought to be directed against disease-relevant antigens.18
For example, most rAbs derived from brain and CSF plasma cell clones in subacute sclerosing panencephalitis bind to measles virus, the cause of subacute sclerosing panencephalitis, and most rAbs derived from CSF plasma cell clones in neuromyelitis optica bind to the disease-associated aquaporin-4 water channel.29–31
Additionally, oligoclonal IgG in CSF of patients with varicella zoster virus vasculopathy is directed against varicella zoster virus antigens.32
Although occasional plasma cells producing anti-EBV antibodies could be present in MS CSF, the absence of anti-EBV reactivity by MS rAbs examined here, which collectively represent the majority of MS patients' clonally expanded plasma cell populations, argues against the role of EBV in MS brain pathogenesis.
Our findings on the rarity of EBV in MS plaques agree with most published studies examining EBV in MS brain. Using PCR amplification, one study detected EBV DNA in about 10% of MS plaques,17
and another in less than 10%.33
Additionally, a higher percentage of EBV-positive samples was present in nonneurologic disease brains (46%) and Parkinson disease brains (42%) as compared to MS (27%) and Alzheimer (25%) brains.17
Multiple studies to detect EBV-specific RNA in MS brains have also been conducted. In situ hybridization (ISH) did not reveal EBV RNA in MS brain,34,35
and another ISH study failed to detect EBV RNA in MS or control tissues, although the authors acknowledged that they were hampered by the poor quality of RNA obtained from the stored brain tissues.36
In contrast, ISH reportedly revealed an abundant presence of EBV RNA in active and chronic-active MS plaques.7
In the latter and the present studies, plaques from patients with primary progressive MS, secondary progressive MS, relapsing-remitting MS, and acute MS were examined (15 plaques by our group and 21 plaques by Serafini et al.7
). Five active and chronic-active plaques analyzed in our study were from 5 patients whose active and chronic-active plaques were reported to contain a high load of EBV.7
These patients (MS079, MS121, MS153, MS154, and MS160) are identified in and in table S1 in reference 7
. Although our group and Serafini et al.7
studied different plaques from these same patients, the plaques examined by both groups contained perivascular cuffs of B- lymphocytes (, table e-2, and table S1 in Serafini et al.7
). Thus, tissue differences are not likely to account for the different results. A more likely explanation for this discrepancy lies in different methods of detection. We used PCR amplification of EBV RNA because of its higher sensitivity and specificity compared to ISH, consistent with a recent study showing that EBV RNA was readily detected by PCR in the same MS samples that were negative by ISH.33
Additionally, PCR amplification allows sequencing of the amplicon and confirmation of specific amplification of the target transcript. Unlike ISH, PCR is highly sensitive, as evidenced by our detection of EBV-specific transcripts in single EBV-infected B cells (including EBER-1 and less abundant EBNA-2, LMP-1, and BFRF-1 transcripts, data not shown) and EBV-positive control tissue.
Our results indicate that the demyelinating events in MS occur largely in the absence of latent or productive EBV infection of B-lymphocytes or plasma cells in MS CNS, and that there is no robust intrathecal anti-EBV antibody production exclusive to MS. Meanwhile, because we did not examine the titer of anti-EBV antibody or the cytotoxic immune response to EBV in MS or control patients, our findings do not contradict the considerable seroepidemiologic data that link EBV infection with an increased risk of developing MS,3–5
or an association of the peripheral anti-EBV antibody titer and HLA-DRB1*15 haplotype with the MRI lesion load and MS brain atrophy.37,38
Overall, latent or productive EBV infection is not present in MS brain or CSF, and there is no specific intrathecal anti-EBV antibody response in MS.