Sven Haahr

Altered CD8+ T cell responses to selected Epstein-Barr virus immunodominant epitopes in patients with multiple sclerosis.

An increased frequency of antiviral CD8+ T cells is seen in chronic viral infections. During herpes virus infections the expanded CD8+ T cells are thought to control the reactivation of the latent infection. Because multiple sclerosis (MS), a presumed autoimmune disease of the central nervous system, has been associated with a late Epstein–Barr virus (EBV) infection, we wished to examine whether the CD8+ T cell response to EBV epitopes differed between MS patients and healthy controls. Here we report an increased frequency of CD8+ T cells responding to EBV epitopes from nuclear antigen 3 A (HLA‐A2/CLG) and latent membrane protein 2 (HLA‐B7/RPP) in MS patients. Noticeably, the altered CD8+ T cell response occurred to some but not all EBV epitopes and did not reach the high level seen during acute infection. The responses towards two immunodominant epitopes from human cytomegalovirus (HCMV) were similar in MS patients and normal controls. Together, our data demonstrate the presence of an increased frequency of CD8+ T cells reacting with two epitopes from EBV in patients with MS. The altered response to only two of the tested EBV epitopes would be consistent with the presence of cross‐reactive epitopes.

A role of late Epstein–Barr virus infection in multiple sclerosis

Objective – To assess Epstein–Barr virus (EBV) seroconversion in a high multiple sclerosis (MS) prevalence area and to evaluate the recall of diagnosed infectious mononucleosis in MS patients.

The implications of Epstein‐Barr virus in multiple‘ sclerosis ‐ a review

The objective of this article is to bring together knowledge about Epstein‐Barr virus (EBV) in relation to multiple sclerosis (MS) in order to evaluate its implications in this disease. All MS patients are EBV seropositive, but EBV is not normally detected in the brain. EBV can explain many of the epidemiological dogmas known in MS. In addition, other studies point towards the involvement of EBV in MS. Despite this, other co‐actors seem also to be involved. We still need to know whether EBV may be an initiating factor in MS or whether it is a factor in the pathogenesis. Possible ways of EBV involvement are discussed: direct involvement, an autoimmune inducing factor or a transactivating factor. A current treatment study of MS patients with a specific herpes antiviral drug may add further information to the etiology and pathogenesis of MS.

A Putative New Retrovirus Associated with Multiple Sclerosis and the Possible Involvement of Epstein-Barr Virus in This Diseasea

Since tropical spastic paraparesis in 1985 was found to be associated with HTLV-I infection, it has been suggested that a retrovirus might be involved in multiple sclerosis (MS). Our group has studied long-term cultures of cerebrospinal fluid cells and peripheral blood mononuclear cells from MS patients and controls with the purpose of elucidating the possible involvement of a retrovirus in MS. For an extended period electron microscopical analysis (EM) of T-cell lines, derived from MS patients and controls and cultured for 4 weeks was performed. In two cultures obtained 8 months apart from a patient with progressive MS, retrovirus-like particles were observed in 1-2% of the cells examined. Recently a B-lymphoblastoid cell line (LCL) producing retrovirus-like particles and EBV was established from a 30-year-old male patient with a chronic progressive myelopathy, clinically resembling multiple sclerosis. Similar cell lines have now been established from two MS patients. The retrovirus-like particles produced by the LCL have been purified by gradient ultracentrifugation. In the purified material reverse transcriptase assays are clearly positive in the gradients where EM shows retrovirus-like particles. Antigen characterization, nucleic acid sequence analysis and antibody studies are now being performed. The retrovirus found is definitively different from other known human retroviruses. It has previously been found that 100% of patients with MS have antibodies against EBV, in contrast to controls where only 86-95% have antibodies against this virus. Previous epidemiological studies have pointed toward a post-pubertal primary EBV infection as an important event in the induction of MS disease. These studies have now been substantiated by our group. Though it is still unknown whether EBV infection is a prerequisite for development of MS or whether the 100% EBV seropositivity is a consequence of the MS disease, we have put forward the hypothesis that the etiological agent for development of MS and MS-like diseases is a new hitherto uncharacterized retrovirus, whereas development of neurologic disease is related to or even dependent on a delayed infection with a virus from the herpes group, most likely EBV. This dual infection hypothesis has been analyzed and was found to be in accordance with the most consistent epidemiological characteristics of MS. We have previously, also from epidemiological data, negated retroviruses, behaving as the known human retroviruses, as an independent cause of MS.

Is Multiple Sclerosis Caused by a Dual Infection with Retrovirus and Epstein-Barr Virus?

Although the etiology of multiple sclerosis is as yet unknown, epidemiological observations strongly point toward one or more infectious agent(s) being involved in the disease. In recent years some studies have indicated involvement of retrovirus in multiple sclerosis (MS). However, an intrafamilial epidemiological study revealed that MS and the known human retroviruses had a divergent epidemiology. Some studies have shown the association of Epstein-Barr virus (EBV) with MS and one recent study revealed dual infection by retrovirus and EBV in a cell line established from a patient with an MS-like disease. Our hypothesis for the development of MS and MS-like diseases is that a hitherto uncharacterized retrovirus is the etiological agent, but development of neurologic disease is related to or even dependent on a delayed EBV infection. The dual infection hypothesis is analyzed and found to be consistent with the epidemiological characteristics of MS.

The significance of Epstein—Barr virus seropositivity in multiple sclerosis patients?

The objective of this study was to evaluate and investigate the significance of the previously found 100% seropositivity toward Epstein–Barr virus (EBV) found in multiple sclerosis (MS) patients in contrast to healthy controls. Using a commercially available ELISA‐test (Biotest), which differentiates infections with EBV into previous infections, primary infections, reactivated infections and no previous infection, we found 137 of 138 MS patients and 124 of 138 healthy controls seropositive. A primary infection in 4 of the 124 EBV seropositive healthy controls in contrast to no primary infections in the MS EBV seropositive group was significant (P=0.049652, Fishers exact test). This may be suggestive of a lack of primary infections in MS patients, and thus strengthens the idea that MS patients are infected with EBV before development of MS. Further studies are in progress to analyse whether EBV infection is a prerequisite for the development of this disease.

Presence of Epstein–Barr virus and human herpesvirus 6B DNA in multiple sclerosis patients: associations with disease activity

Objective –  To assess the presence of Epstein–Barr virus (EBV) and human herpesvirus 6B (HHV‐6B) DNA in saliva and plasma from multiple sclerosis (MS) patients enrolled in a randomized, double‐blind, placebo‐controlled valacyclovir treatment study.

A single subtype of Epstein–Barr virus in members of multiple sclerosis clusters

Objectives – Epidemiological studies strongly indicate an infectious involvement in multiple sclerosis (MS). Epstein–Barr virus (EBV), to which all multiple sclerosis patients are seropositive, is also interesting from an epidemiological point of view. We have reported a cluster of MS patients with 8 members from a small Danish community called Fjelsø. To further evaluate the role of EBV in MS we have investigated the distribution of EBV subtypes in cluster members and in control cohorts. Materials and methods – Blood mononuclear cells were isolated from cluster members, unrelated MS patients, healthy controls, including healthy schoolmates to the Fjelstø cluster patients and finally from persons with autoimmune diseases in order to investigate the number of 39 bp repeats in the EBNA 6‐coding region in the EBV seropositive individuals. Results – We observed a preponderance of the subtype with 3 39 bp repeats in the EBNA 6‐coding region both in the MS patients and the healthy controls. In the Fjelsø cluster all 8 cluster members were harbouring this subtype, which is significantly different from the finding in healthy controls (n = 16), which include 8 schoolmates to the cluster members and 8 randomly selected healthy persons (Fischers exact test P=0.0047), and also compared to all non‐clustered individuals studied (P=0.017). Conclusion – Infection with the same subtype of EBV links together the 8 persons from the Fjelso cluster who later developed MS. This finding adds to the possibility that development of MS is linked to infection with EBV.

Retrovirus-like particles in an Epstein-Barr virus-producing cell line derived from a patient with chronic progressive myelopathy

A B‐lymphoblastoid cell line (LCL) of polyclonal origin was isolated from a 30‐year‐old male patient with a chronic progressive myelopathy clinically resembling multiple sclerosis (MS). The LCL expresses Epstein‐Barr virus (EBV) encoded proteins and on transmission electron microscopy (EM) the LCL was shown to produce both EBV particles and retrovirus‐like particles spontaneously. The LCL was negative for human retrovirus (HIV‐I and HTLV‐I) sequences by polymerase chain reaction (PCR). Furthermore the patient was seronegative to these retroviruses including HTLV‐II and HIV‐II. We, therefore, suggest that the LCL is double‐infected with EBV and a hitherto uncharacterized human retrovirus. The possible implications of these two viruses on development of diseases are discussed.

B-lymphoblastoid cell lines from multiple sclerosis patients and a healthy control producing a putative new human retrovirus and Epstein-Barr virus.

On several occasions we have observed retrovirus-like particles (RVLPs) by transmission electron microscopy (EM) of cultured T cells from a patient with MS. Later we established spontaneously formed B-lymphoblastoid cell lines (LCLs) from a patient with an MS-like disease and from another patient with MS who had a reactivated Epstein-Barr virus (EBV) infection. Both LCLs were found by EM to produce RVLP and EBV particles. Reverse transcriptase (RT) assays were positive in purified viral material from both LCLs. To substantiate these findings we initiated an intensified culturing procedure and were able to establish LCLs from 5 out of 21 consecutive MS patients and 1 out of 13 consecutive healthy controls. All LCLs were found to produce both RVLP and EBV particles by EM. Whether the putative new retrovirus(es) and EBV have any causal relationship to MS is still not known, but the findings support this possibility.