The results of two multicenter, open-label studies assessing efficacy, tolerability and safety of protiramer, a high molecular weight synthetic copolymeric mixture, in patients with relapsing-remitting multiple sclerosis.

OBJECTIVE: Two pilot studies were conducted to evaluate safety, tolerability, and efficacy of two doses of Protiramer (TV-5010) in patients with relapsing-remitting multiple sclerosis. BACKGROUND: Both glatiramer acetate and TV-5010 are synthetic copolymers comprised the same four amino acids in a defined molar ratio. TV-5010 has higher average molecular weight than Glatiramer acetate and might be hypothesized that glatiramoids with higher molecular weight might be more immunoreactive than lower molecular weight peptides, thus increasing therapeutic potential and allowing for less frequent dosing. METHODS: In the two separate studies, after a 10 week pretreatment period, TV-5010 was given subcutaneously once weekly at 15 mg and 30 mg for 36 weeks. The primary end point was a reduction in the number of magnetic resonance imaging active lesions (i.e., T1-weigthed gadolinium-enhancing and new T2-weighted lesions) between the pretreatment period and the end of study. RESULTS: Both TV-5010 doses were generally well tolerated. The treatment with TV-5010 at a dose of 15 mg/wk did not show any significant effect. In contrast, in patients treated with at a dose of 30 mg/wk, a significant reduction in the mean number of gadolinium-enhancing (-58.8%; P = 0.0013) and new T2-W (-50%; P = 0.0002) lesions was observed. However, a large decrease in the mean number of both gadolinium-enhancing (-55%) and new T2-W (-40%) lesions during the pretreatment period made difficult the interpretation of the efficacy assessments. CONCLUSIONS: Further studies are needed to confirm these preliminary data on safety and efficacy of TV-5010 at a weekly dose of 30 mg.

Long-term follow up of glatiramer acetate compassionate use in Belgium.

Between June 1995 and November 1998, 228 patients with relapsing-remitting Multiple Sclerosis started treatment with glatiramer acetate (Copaxone) 20 mg once daily in the frame of a "compassionate use" protocol in 15 Belgian centers. Following an average treatment period of 5.8 years, treating neurologists were requested to fill in follow-up forms indicating neurological disability status and side effects during the previous 6 months. These data were available for 134 patients. In this group, the Expanded Disability Status Scale (EDSS) improved in 26.3% of patients. An additional 36.8% of patients remained neurologically stable. The Ambulation Index (AI) showed similar results: 12.5% of patients improved, 50% of patients remained stable, and 37.5% worsened. Only 10% of patients dropped out due to several reasons. The adverse events occurring in the period preceding the follow-up survey were non-serious and consistent with the current product information of glatiramer acetate. Among the 94 patients no longer followed-up in the compassionate program, reasons for lost to follow-up were obtained for 63; most of them (41) had stopped GA treatment or switched to another disease-modifying treatment. Overall these results are very similar to the ones reported in the extension study of the pivotal trial (Johnson et al., 2000), and indicate that patients treated with glatiramer acetate have a better outcome than expected on the basis of the natural course of the disease. Despite limitations of the study design, this report confirms the sustained efficacy of glatiramer acetate in reducing the disease progression in patients with relapsing-remitting multiple sclerosis treated in day-to-day clinical practice.

T cell vaccination in multiple sclerosis patients with autologous CSF-derived activated T cells: results from a pilot study.

Myelin-reactive T cells are considered to play an essential role in the pathogenesis of multiple sclerosis (MS), an autoimmune disease of the central nervous system. We have previously studied the effects of T cell vaccination (TCV), a procedure by which MS patients are immunized with attenuated autologous myelin basic protein (MBP)-reactive T cell clones. Because several myelin antigens are described as potential autoantigens for MS, T cell vaccines incorporating a broad panel of antimyelin reactivities may have therapeutic effects. Previous reports have shown an accumulation of activated T cells recognizing multiple myelin antigens in the cerebrospinal fluid (CSF) of MS patients. We conducted a pilot clinical trial of TCV with activated CD4+ T cells derived from CSF in five MS patients (four RR, one CP) to study safety, feasibility and immune effects of TCV. CSF lymphocytes were cultured in the presence of rIL-2 and depleted for CD8 cells. After 5-8 weeks CSF T cell lines (TCL) were almost pure TCR alpha beta+CD4+ cells of the Th1/Th0 type. The TCL showed reactivity to MBP, MOG and/or PLP as tested by Elispot and had a restricted clonality. Three immunizations with irradiated CSF vaccines (10 million cells) were administered with an interval of 2 months. The vaccinations were tolerated well and no toxicity or adverse effects were reported. The data from this small open-label study cannot be used to support efficacy. However, all patients remained clinically stable or had reduced EDSS with no relapses during or after the treatment. Proliferative responses against the CSF vaccine were observed in 3/5 patients. Anti-ergotypic responses were observed in all patients. Anti-MBP/PLP/MOG reactivities remained low or were reduced in all patients. Based on these encouraging results, we recently initiated a double-blind placebo-controlled trial with 60 MS patients to study the effects of TCV with CSF-derived vaccines in early RR MS patients.

T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls.

Myelin proteins, including myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) are candidate autoantigens in MS. It is not clear whether MS patients show a predominant reactivity to one or several myelin antigens. We evaluated the IFN-gamma production induced by MBP and MOG and selected MBP-, MOG- and PLP-peptides in MS patients and healthy controls using the IFN-gamma ELISPOT assay. Most MS patients and healthy controls showed a heterogeneous anti-myelin T-cell reactivity. Interestingly in MS patients a positive correlation was found between the anti-MOG and anti-MBP T-cell responses. No myelin peptide was preferentially recognized among the peptides tested (MBP 84-102, 143-168, MOG 1-22, 34-56, 64-86, 74-96, PLP 41-58, 184-199, 190-209). In addition the frequency of IL2R+ MBP reactive T-cells was significantly increased in blood of MS patients as compared with healthy subjects, indicating that MBP reactive T-cells exist in an in vivo activated state in MS patients. Most of the anti-MBP T-cells were of the Th1-type because reactivity was observed in IFN-gamma but not in IL-4 ELISPOT-assays. Using Th1 (IL-12) and Th2 (IL-4) promoting conditions we observed that the cytokine secretion pattern of anti-MBP T-cells still is susceptible to alteration. Our data further indicate that precursor frequency analysis of myelin reactive T-cells by proliferation-based assays may underestimate the true frequency of myelin specific T-cells significantly.

Myelin reactive T cells after T cell vaccination in multiple sclerosis: cytokine profile and depletion by additional immunizations.

Pathogenic autoreactive T cells can be targeted by T cell vaccination (TCV), a procedure in which patients are immunized with autologous attenuated pathogenic T cells. We reported previously that TCV with myelin basic protein (MBP) reactive T cell clones in multiple sclerosis (MS) patients induced T cell immune responses, resulting in a clonal depletion of MBP reactive T cells in all patients. Five years after TCV, MBP reactive T cells were observed in five out of nine MS patients. These clones had a different clonal origin from those isolated before vaccination. We have studied the cytokine profile, cytotoxicity and epitope specificity of these reappearing clones. Our data indicate that the clones express similar effector functions as those isolated before TCV, suggesting that they also could play a pathogenic role in the disease. We demonstrated that these clones can be depleted by an additional sequence of immunizations. These findings may be relevant to other T cell targeted immunotherapies for MS and other autoimmune diseases.

Myelin reactive T cells in the autoimmune pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination. Although it is widely accepted that demyelination in MS results from an active inflammatory process, the cause of the inflammation is still not completely resolved. Findings in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and observations in human MS have led to the hypothesis that MS is an autoimmune disease mediated by autoreactive T cells with specificity for myelin antigens. The identity of the brain antigen(s) which is (are) the primary target(s) of the autoimmune process is not known, but current evidence indicates that myelin basic protein (MBP) is a likely candidate. In this paper we will overview some of the experimental evidence suggesting that MBP reactive T cells hold a central position in the pathogenesis of MS, and discuss some of the currently tested therapeutic strategies in MS which are directed towards the pathogenic MBP reactive T cells. Although there appears to be no direct correlation between anti-MBP T cell responses and clinical disease activity, some recent observations suggest that monitoring of anti-MBP T cell responses could be helpful to study immunological efficacy of experimental immunotherapies in MS.

Risks of multiple sclerosis in relatives of patients in Flanders, Belgium.

OBJECTIVES: To calculate age adjusted risks for multiple sclerosis in relatives of Flemish patients with multiple sclerosis. METHODS: Lifetime risks were calculated using the maximum likelihood approach. RESULTS: Vital information was obtained on 674 probands with multiple sclerosis in Flanders and a total of their 26225 first, second, and third degree relatives. Full medical information to allow documentation of multiple sclerosis status was available for 21351 (81.4%) relatives. The age adjusted risk for parents was 1.61 (SEM 0.35)%, for siblings 2.10 (SE 0.36)%, and for children 1.71 (SEM 0.70)%. For aunts and uncles, the risk was 0.66 (SEM 0.13)%. CONCLUSIONS: The risk for first degree relatives of patients with multiple sclerosis in Flanders is increased 10-fold to 12-fold; for second degree relatives, it is increased threefold. This information can be used for risk counselling in families and provides additional support for the role of more than one locus contributing to the susceptibility of multiple sclerosis.

T cell vaccination: clinical application in autoimmune diseases.

T cell responses to myelin basic protein (MBP) are implicated to play an important role in the pathogenesis of multiple sclerosis (MS). These MBP autoreactive T cells are found to undergo in vivo activation and clonal expansion in patients with MS. They accumulate in the brain compartment and may reside in the brain lesions of patients with MS. As MBP-reactive T cells potentially hold a central position in initiation and perpetuation of the brain inflammation, specific immune therapies designed to deplete them may improve the clinical course of the disease. We review here the recent application of T cell vaccination in patients with MS to deplete circulating MBP-reactive T cells. The results of our phase I clinical trial indicate that T cell vaccination with inactivated MBP autoreactive T cells induces specific regulatory T cell network of the host immune system to deplete circulating MBP-reactive T cells in a clonotype-specific fashion. The immunity induced by T cell vaccination is clonotype specific and long-lasting. Our longitudinal clinical evaluation further suggests a moderately lower rate of clinical exacerbation, disability score, and brain lesions (measured by magnetic resonance imaging) in vaccinated patients than in matched controls. Our study should encourage further investigation on the treatment efficacy of T cell vaccination and further improvement for its clinical administration in other human autoimmune diseases. This review discusses the immune regulation and therapeutic administration of T cell vaccination in human autoimmune diseases, exemplified by our recent T cell vaccination trial in MS.

Vaccination with autoreactive T cell clones in multiple sclerosis: overview of immunological and clinical data.

Although the etiology and pathogenesis of Multiple Sclerosis (MS) remain elusive, accumulating evidence indicates that MS is a chronic inflammatory disease with an autoimmune component, mediated by autoreactive T lymphocytes specific for myelin antigens. The triggering T cell autoantigen has not been identified yet, but recent immunological studies in MS and parallel experiments in experimental allergic encephalomyelitis (EAE), the animal model of MS, have indicated that myelin basic protein (MBP) can be considered as one of the major candidate autoantigens in the pathogenesis of the disease. Based on these observations, several therapeutic strategies have been developed aimed at the specific elimination or inactivation of MBP reactive T cells in MS. One of these approaches involves the immunization of MS patients with autologous attenuated autoreactive T cells to induce an immune response specifically targeted at these autoreactive T cells. This method, termed T cell vaccination, has been shown to prevent and treat EAE. We have recently conducted a pilot trial of T cell vaccination in a limited group of MS patients to evaluate the immunological responses to the injected cells. The data obtained indicate that this type of vaccination induces an effective anti-clonotypic T cell response leading to a specific depletion of circulating MBP reactive T cells. Preliminary data on the clinical effects are promising, encouraging further clinical trials.

Depletion of myelin-basic-protein autoreactive T cells by T-cell vaccination: pilot trial in multiple sclerosis.

In a pilot trial, eight patients with multiple sclerosis were matched to control patients and received T-cell vaccination with irradiated T cells reactive to myelin basic protein (MBP) to deplete circulating MBP-reactive T cells. In the 2 years before and after vaccination, exacerbations decreased in five vaccinated patients (numbers 1, 2, 6-8) with relapsing-remitting disease from sixteen to three, respectively, and from twelve to ten in their matched controls. Magnetic resonance imaging showed a mean 8.0% increase in brain lesion size in the vaccinated patients compared with a 39.5% increase in the controls. Lesions and/or relapses worsened in three cases after vaccination in association with reappearance of circulating MBP-reactive T cells.

Increased frequency of gamma delta T cells in cerebrospinal fluid and peripheral blood of patients with multiple sclerosis. Reactivity, cytotoxicity, and T cell receptor V gene rearrangements.

Infiltrating gamma delta T cells are potentially involved in the central nervous system demyelination in multiple sclerosis (MS). To further study this hypothesis, we analyzed the frequency and functional properties of gamma delta T cells in peripheral blood (PB) and paired cerebrospinal fluid (CSF) of patients with MS and control subjects, including patients with other neurologic diseases (OND) and healthy individuals. The frequency analysis was performed under limiting dilution condition using rIL-2 and PHA. After PHA stimulation, a significantly increased frequency of gamma delta T cells was observed in PB (14.7 x 10(-4)) and in CSF (15.8 x 10(-4)) of MS patients as compared with 4.3 x 10(-4) in PB and 3.9 x 10(-4) detected in CSF of patients with OND. The frequency was represented equally in OND patients and normal individuals. Similarly, the IL-2-responsive gamma delta T cells occurred at a higher frequency in PB of control subjects (1.1 x 10(-4)) in OND patients and 1.5 x 10(-4) in normal individuals). Forty-three percent (13 of 30) of the gamma delta T cell clones isolated from PB and CSF of MS patients responded to heat shock protein (HSP70) but not HSP65, whereas only 2 of 30 control gamma delta T cell clones reacted to the HSP. The majority of the gamma delta T cell clones were able to induce non-MHC-restricted cytolysis of Daudi cells. All clones displayed a substantial reactivity to bacterial superantigens staphylococcal enterotoxin B and toxic shock syndrome toxin-1, irrespective of their gamma delta V gene usage. Furthermore, the gamma delta T cell clones expressed predominantly TCRDV2 and GV2 genes (26 of 35 clones), whereas the clones derived from CSF of MS patients expressed either DV1 or DV2 genes. The obtained gamma delta clones, in general, represented rather heterogeneous clonal origins, even though a predominant clonal origin was found in a set of 10 gamma delta clones derived from one patient with MS. The present study provides new evidence supporting a possible role of gamma delta T cells in the secondary inflammatory processes in MS.

Clonal expansion of myelin basic protein-reactive T cells in patients with multiple sclerosis: restricted T cell receptor V gene rearrangements and CDR3 sequence.

Myelin basic protein (MBP)-reactive T cells are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In some patients with MS, these autoreactive T cells display a limited heterogeneity in their epitope recognition and T cell receptor (TCR) variable (V) gene usage. These individual-dependent properties of MBP-reactive T cells have led to the speculation that they may represent clonal expansion in vivo in some MS patients. In the present study, 51 MBP-reactive T cell clones derived from patients with MS and healthy individuals were examined for their epitope recognition and the TCR V alpha and V beta gene rearrangements. The V gene junctional region sequences of identified alpha and beta genes were further analyzed to probe their clonal origins, as the sequences are unique for individual clones. Our data showed that 26 clones derived from nine patients with MS shared a predominant reactivity to the immunodominant regions of MBP, 84-102, 110-129 and 143-168, and used various TCR V alpha and V beta rearrangements. The V gene usage of the clones was restricted to certain V alpha V beta combination(s) in a given MS patient, but varied among different patients. The sequence analysis revealed that the clones generated from a given patient shared a limited or a single junctional region sequence pattern(s), indicating their oligoclonal or monoclonal origin(s). In contrast, 25 MBP-reactive T cell clones derived from normal individuals exhibited unfocused epitope recognition and V gene usage. Thus, the limited heterogeneity of MBP-reactive T cells in their structural and functional characteristics reflects their clonal expansion in vivo in some patients with MS.

Focal leptomeningeal MR enhancement along the chiasm as a presenting sign of multiple sclerosis.

We demonstrate focal leptomeningeal enhancement along the chiasm on MRI in a 28-year-old man presenting with blurred vision and bitemporal visual field defects. The diagnosis of clinically definite multiple sclerosis was confirmed by laboratory investigations and brain MR findings.

HLA and T-cell receptor polymorphisms in Belgian multiple sclerosis patients: no evidence for disease association with the T-cell receptor.

There are compelling data to indicate that the susceptibility to multiple sclerosis (MS) is inherited, at least in part. Particular HLA genotypes may be associated with MS and recently also polymorphisms in the T-cell receptor (TCR) genes have been reported to correlate with the disease; however, these data have been difficult to confirm. We investigated the TCRA and TCRB chain genes of HLA-typed Belgian CP MS patients employing four DNA restriction fragment length polymorphisms (RFLPs) detected with TCR constant (TCRAC1, TCRBC2) and variable (TCRBV8, TCRBV11) gene segments. Similar frequencies in patients and controls were observed for all RFLPs studied. Although the HLA DR2 genotype was significantly associated with MS, no interactive effects were seen with MS, DR2, TCRAC1, TCRBC2 and TCRBV alleles. We conclude that, while a clear association with HLA DR2 is observed, little convincing evidence exists for an association of CP MS with RFLPs of the TCRA or TCRB chain genes.

MHC-restricted depletion of human myelin basic protein-reactive T cells by T cell vaccination.

Activated autoreactive T cells are potentially pathogenic and regulated by clonotypic networks. Experimental autoimmune diseases can be treated by inoculation with autoreactive T cells (T cell vaccination). In the present study, patients with multiple sclerosis were inoculated with irradiated myelin basic protein (MBP)-reactive T cells. T cell responses to the inoculates were induced to deplete circulating MBP-reactive T cells in the recipients. Regulatory T cell lines isolated from the recipients inhibited T cells used for vaccination. The cytotoxicity of the CD8+ T cell lines was restricted by major histocompatibility antigens. Thus, clonotypic interactions regulating autoreactive T cells in humans can be induced by T cell vaccination.

JC virus and multiple sclerosis: a refutation?

Polyomavirus JC (JCV) has been implicated in the etiology of multiple sclerosis (MS), because it causes progressive multifocal leukoencephalopathy (PML), a multifocal demyelinating disease with many microscopical similarities to MS. During childhood, the virus establishes a latent infection in the kidneys, which can be reactivated in immunocompromised patients. During reactivation, the virus is shed in the urine. The kidney is the only known site of latent infection and reactivation. Therefore, excretion of the virus in the urine of MS patients is to be expected, if reactivated JCV is involved in the etiology of MS. We studied urine samples of 53 patients with definitive MS and of 53 controls matched for age and sex. We found no evidence of active JCV infection in MS. The hypothesis of a polyomaviral etiology of MS is not supported by the results of this study.

Myelin basic protein-specific T lymphocytes in multiple sclerosis and controls: precursor frequency, fine specificity, and cytotoxicity.

A panel of 90 myelin basic protein (MBP)-specific T-cell lines were derived from peripheral blood of eight patients with multiple sclerosis and four normal subjects. The precursor frequency of MBP-reactive T cells in peripheral blood mononuclear cells ranged from 10(-7) to 9 x 10(-7) (mean, 6.7 x 10(-7)) in the group of patients with multiple sclerosis and from 0.5 x 10(-7) to 9.8 x 10(-7) (mean, 5.6 x 10(-7)) in the control subjects. This difference between the two groups was not statistically significant (p greater than 0.1). These T-cell lines expressed exclusively CD3+CD4+CD8- phenotypes and were restricted predominantly by HLA-DR molecules. When tested with fragments and synthetic peptides of human MBP, these MBP-specific T-cell lines (45 lines for each group) displayed a limited heterogeneous pattern with a biased recognition to peptide 84-102 and the C-terminal peptide 149-171. The reactivity to the 84-102 region of MBP was associated with the HLA-DR2, DRw15 (DRw15,2) haplotype, whereas the recognition to peptide 149-171 did not correlate with a particular HLA-DR allele(s). Furthermore, the majority of T-cell lines (greater than 75%) were found to exhibit substantial cytotoxic activity against MBP-coated target cells, but showing no significant difference between these two groups. This MBP-dependent cytotoxicity was not associated with epitope specificities of the T-cell lines tested.

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.

Antibodies to myelin basic protein and measles virus in multiple sclerosis: precursor frequency analysis of the antibody producing B cells.

Antibody-producing B lymphocytes were polyclonally activated and transformed, by Epstein-Barr virus (EBV), into multiple B lymphoblastoid cell lines in a microculture system. The frequencies of B precursor cells producing antibodies to myelin basic protein (MBP) and measles virus were analyzed in peripheral blood of patients with multiple sclerosis (MS) and control subjects. Measles virus-specific B cells were detected at a significantly higher frequency in MS patients (n = 10, P less than 0.005) than patients with other neurological diseases (n = 10) and normal subjects (n = 10). In contrast, the frequencies of B cells producing anti-MBP antibodies and natural antibodies did not differ statistically among the three groups tested (P greater than 0.05). In addition, the anti-MBP antibodies produced by a panel of stable B cell lines obtained were found to react selectively with an epitope(s) within the C-terminal half fragment 90-171 of the human MBP molecule. In our experiments, no antibody cross-reactivity between MBP and measles virus could be detected in a total of 2760 B cell cultures.

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients.

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.