Epstein-Barr Virus in Multiple Sclerosis: Theory and Emerging Immunotherapies.

New treatments for multiple sclerosis (MS) focused on B cells have created an atmosphere of excitement in the MS community. B cells are now known to play a major role in disease, demonstrated by the highly impactful effect of a B cell-depleting antibody on controlling MS. The idea that a virus may play a role in the development of MS has a long history and is supported mostly by studies demonstrating a link between B cell-tropic Epstein-Barr virus (EBV) and disease onset. Efforts to develop antiviral strategies for treating MS are underway. Although gaps remain in our understanding of the etiology of MS, the role, if any, of viruses in propagating pathogenic immune responses deserves attention.

Epstein-Barr virus-specific T cell therapy for progressive multiple sclerosis.

BACKGROUND: Increasing evidence indicates a role for EBV in the pathogenesis of multiple sclerosis (MS). EBV-infected autoreactive B cells might accumulate in the CNS because of defective cytotoxic CD8+ T cell immunity. We sought to determine the feasibility and safety of treating progressive MS patients with autologous EBV-specific T cell therapy. METHODS: An open-label phase I trial was designed to treat 5 patients with secondary progressive MS and 5 patients with primary progressive MS with 4 escalating doses of in vitro-expanded autologous EBV-specific T cells targeting EBV nuclear antigen 1, latent membrane protein 1 (LMP1), and LMP2A. Following adoptive immunotherapy, we monitored the patients for safety and clinical responses. RESULTS: Of the 13 recruited participants, 10 received the full course of T cell therapy. There were no serious adverse events. Seven patients showed improvement, with 6 experiencing both symptomatic and objective neurological improvement, together with a reduction in fatigue, improved quality of life, and, in 3 patients, reduced intrathecal IgG production. All 6 patients receiving T cells with strong EBV reactivity showed clinical improvement, whereas only 1 of the 4 patients receiving T cells with weak EBV reactivity showed improvement (P = 0.033, Fisher's exact test). CONCLUSION: EBV-specific adoptive T cell therapy was well tolerated. Clinical improvement following treatment was associated with the potency of EBV-specific reactivity of the administered T cells. Further clinical trials are warranted to determine the efficacy of EBV-specific T cell therapy in MS. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12615000422527. FUNDING: MS Queensland, MS Research Australia, Perpetual Trustee Company Ltd., and donations from private individuals who wish to remain anonymous.

Increased constitutive activation of NF-κB p65 (RelA) in peripheral blood cells of patients with progressive multiple sclerosis.

The NF-κB signalling pathway plays an important role in controlling cellular immune responses, inflammation and apoptosis. In multiple sclerosis (MS), there is evidence of dysregulation of NF-κB signalling in patients with a relapsing-remitting disease course, but thus far there is little information on whether it is also dysregulated in patients with progressive disease. We hypothesised that patients with progressive MS would have more activation of NF-κB than relapsing-remitting MS patients. Using several different methods, we showed that there was more nuclear translocation of p65 in cells from progressive MS patients, particularly in T cells and monocytes. In addition, the amount of p65 translocated to the nucleus in cells of patients with progressive MS was not increased upon non-specific activation of the cells with the mitogen Con A. These results suggest that NF-κB dysregulation occurs in patients with progressive MS patients, as well as those with relapsing-remitting MS.

Epstein-Barr virus and multiple sclerosis: potential opportunities for immunotherapy.

Multiple sclerosis (MS) is a common chronic inflammatory demyelinating disease of the central nervous system (CNS) causing progressive disability. Many observations implicate Epstein-Barr virus (EBV) in the pathogenesis of MS, namely universal EBV seropositivity, high anti-EBV antibody levels, alterations in EBV-specific CD8(+) T-cell immunity, increased spontaneous EBV-induced transformation of peripheral blood B cells, increased shedding of EBV from saliva and accumulation of EBV-infected B cells and plasma cells in the brain. Several mechanisms have been postulated to explain the role of EBV in the development of MS including cross-reactivity between EBV and CNS antigens, bystander damage to the CNS by EBV-specific CD8(+) T cells, activation of innate immunity by EBV-encoded small RNA molecules in the CNS, expression of αB-crystallin in EBV-infected B cells leading to a CD4(+) T-cell response against oligodendrocyte-derived αB-crystallin and EBV infection of autoreactive B cells, which produce pathogenic autoantibodies and provide costimulatory survival signals to autoreactive T cells in the CNS. The rapidly accumulating evidence for a pathogenic role of EBV in MS provides ground for optimism that it might be possible to prevent and cure MS by effectively controlling EBV infection through vaccination, antiviral drugs or treatment with EBV-specific cytotoxic CD8(+) T cells. Adoptive immunotherapy with in vitro-expanded autologous EBV-specific CD8(+) T cells directed against viral latent proteins was recently used to treat a patient with secondary progressive MS. Following the therapy, there was clinical improvement, decreased disease activity on magnetic resonance imaging and reduced intrathecal immunoglobulin production.

Deficiency of CD8+ effector memory T cells is an early and persistent feature of multiple sclerosis.

BACKGROUND: Patients with multiple sclerosis (MS) have a deficiency of circulating CD8+ T cells, which might impair control of Epstein-Barr virus (EBV) and predispose to MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. Based on the expression of CD45RA and CD62L, CD4+ T cells and CD8+ T cells can be subdivided into four subsets with distinct homing and functional properties, namely: naïve, central memory, effector memory (EM) and effector memory re-expressing CD45RA (EMRA) cells. OBJECTIVE: Our aim was to determine which memory subsets are involved in the CD8+ T cell deficiency and how these relate to clinical course. METHODS: We used flow cytometry to analyze the memory phenotypes of T cells in the blood of 118 MS patients and 112 healthy subjects. RESULTS: MS patients had a decreased frequency of EM (CD45RA(-)CD62L(-)) and EMRA (CD45RA(+)CD62L(-)) CD8+ T cells, which was present at the onset of disease and persisted throughout the clinical course. The frequencies of CD4+ EM and EMRA T cells were normal. CONCLUSION: Deficiency of effector memory CD8+ T cells is an early and persistent feature of MS and might underlie the impaired CD8+ T cell control of EBV.

Epstein-Barr virus-specific adoptive immunotherapy for progressive multiple sclerosis.

Defective control of Epstein-Barr virus (EBV) infection by cytotoxic CD8(+) T cells might predispose to multiple sclerosis (MS) by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. We have treated a patient with secondary progressive MS with in vitro-expanded autologous EBV-specific CD8(+) T cells directed against viral latent proteins. This adoptive immunotherapy had no adverse effects and the patient showed clinical improvement with reduced disease activity on magnetic resonance imaging and decreased intrathecal immunoglobulin production. This is the first report of the use of EBV-specific adoptive immunotherapy to treat MS or any other autoimmune disease.

The physical anthropometry, lifestyle habits and blood pressure of people presenting with a first clinical demyelinating event compared to controls: the Ausimmune study.

INTRODUCTION: Lifestyle factors prior to a first clinical demyelinating event (FCD), a disorder often preceding the development of clinically definite multiple sclerosis (MS), have not previously been examined in detail. Past tobacco smoking has been consistently associated with MS. METHODS: This was a multicentre incident case-control study. Cases (n = 282) were aged 18-59 years with an FCD and resident within one of four Australian centres (from latitudes 27°S to 43°S), from 1 November 2003 to 31 December 2006. Controls (n = 558) were matched to cases on age, sex and study region, without CNS demyelination. Exposures measured included current and past tobacco and marijuana, alcohol and beverage use, physical activity patterns, blood pressure and physical anthropometry. RESULTS: A history of smoking ever was associated with FCD risk (AOR 1.89 (95%CL 1.82, 3.52)). Marijuana use was not associated with FCD risk after adjusting for confounders such as smoking ever but the estimates were imprecise because of a low prevalence of use. Alcohol consumption was common and not associated with FCD risk. No case-control differences in blood pressure or physical anthropometry were observed. CONCLUSIONS: Past tobacco smoking was positively associated with a risk of FCD but most other lifestyle factors were not. Prevention efforts against type 2 diabetes and cardiovascular disease by increasing physical activity and reducing obesity are unlikely to alter MS incidence, and more targeted campaigns will be required.

Sun exposure over a lifetime in Australian adults from latitudinally diverse regions.

Spatio-temporal patterns in sun exposure underlie variations in skin cancer incidence and vitamin D deficiency, indicate effectiveness of sun protection programs and provide insights into future health risks. From 558 adults across four regions of Australia (Brisbane (27°S), Newcastle (33°S), Geelong and the Western Districts of Victoria (37°S) and Tasmania (43°S)), we collected: self-report data on time-in-the-sun from age 6 years; natural skin color and ethnicity; silicone skin casts (for cumulative skin damage); and serum for vitamin D status. Ambient ultraviolet radiation (UVR) at the location of residence, with time-in-the-sun, was used to calculate a "UVR dose" for each year of life. Individuals maintained their ranking compared to their peers for time-in-the-sun in summer compared to winter and across ages (Spearman rho 0.24-0.84, all P < 0.001). Time-in-the-sun decreased with age in all birth cohorts, and over calendar time. Summer time-in-the-sun increased with increasing latitude (P < 0.001). Seasonal variation in vitamin D status had greater amplitude and vitamin D deficiency increased with increasing latitude. Temporal patterns are consistent with effectiveness of sun protection programs. Higher relative time-in-the-sun persists from childhood through adulthood. Lower summer time-in-the-sun in the warmest location may have implications for predictions of UVR-related health risks of climate change.

Adherence to MRI protocol consensus guidelines in multiple sclerosis: an Australian multi-centre study.

INTRODUCTION: Multiple sclerosis (MS) is a debilitating disease that causes significant morbidity within a young demographic. Diagnostic guidelines for MS have evolved, and imaging has played an increasingly important role in diagnosis over the last two decades. For imaging to contribute to diagnosis in a meaningful way, it must be reproducible. Consensus guidelines for MRI in MS exist to define correct sequence type and imaging technique, but it is not clear to what extent they are followed. This study reviewed MRI studies performed on Australian individuals presenting with a first clinical diagnosis of central nervous system demyelination (FCD) for adherence to published guidelines and discussed practical implementation of MS guidelines in light of recent updates. METHODS: The Ausimmune study was a prospective case control study of Australian participants presenting with FCD from 2003 to 2006. Baseline cranial and spinal cord MRI studies of 226 case participants from four separate Australian regions were reviewed. MRI sequences were classified according to anatomical location, slice plane, tissue weighting and use of gadolinium-containing contrast media. Results were compared with the 2003 Consortium of Multiple Sclerosis Centres MRI protocol for the diagnosis of MS. RESULTS: The composition of core cranial MRI sequences performed varied across the 226 scans. Of the studies, 91% included sagittal fluid attenuated inversion recovery (FLAIR) sequences. Cranial axial T2-weighted, axial FLAIR and axial proton density-weighted sequences were performed in 88%, 60% and 16% (respectively) of scans. Only 25% of the studies included a T1-weighted contrast-enhanced sequence. Concordance with the guidelines in all sequences was very low (2). CONCLUSION: Only a small number of MRI investigations performed included all of the sequences stipulated by consensus guidelines. This is likely due to poor awareness in the imaging community of the guidelines and the rationale behind certain sequences. Radiologists with a sub-speciality interest in neuroradiology should take ownership of this issue and ensure that recommended imaging guidelines are followed.

Interleukin-6 gene promoter-572 C allele may play a role in rate of disease progression in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease affecting the central nervous system. Although the exact pathogenesis of MS is unknown, it is generally considered to be an autoimmune disease, with numerous genetic and environmental factors determining disease susceptibility and severity. One important mediator of immune responses and inflammation is interleukin-6 (IL-6). Previously, elevated levels of IL-6 in mononuclear cells in blood and in brain tissue from MS patients have been reported. Various polymorphisms in the promoter region of the IL6 gene have also been linked with IL-6 protein levels. In MS, several small studies have investigated whether two IL6 promoter polymorphisms (-597 G>A and -174 G>C) correlate with MS susceptibility, but with varying results. In the present study, we analyzed these polymorphisms, together with an additional polymorphism (-572 G>C) in 279 healthy controls and 509 patients with MS. We found no significant differences between MS patients and healthy controls for the different -597 or -174 IL6 promoter alleles or genotypes. There was a slight reduction in the percentage of individuals with MS who carried a C allele at position -572, although this was not significant after correction for multiple comparisons. Interestingly, however, the -572 C allele showed a significant correlation with the MS severity score, suggesting a possible role in disease progression.

Decreased CD8+ T cell response to Epstein-Barr virus infected B cells in multiple sclerosis is not due to decreased HLA class I expression on B cells or monocytes.

BACKGROUND: Patients with multiple sclerosis (MS) have a decreased frequency of CD8+ T cells reactive to their own Epstein-Barr virus (EBV) infected B cells. We have proposed that this might predispose to the development of MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. The decreased CD8+ T cell response to EBV results from a general CD8+ T cell deficiency and also a decreased proportion of EBV-specific T cells within the total CD8+ T cell population. Because decreased HLA class I expression on monocytes and B cells has been reported in MS and could influence the generation and effector function of EBV-specific CD8+ T cells, the present study was undertaken to measure the expression of HLA molecules on B cells and monocytes in patients with MS. METHODS: We used flow cytometry to determine the proportions of T cells, natural killer cells, B cells and monocytes in peripheral blood mononuclear cells (PBMC) and to quantify the expression of HLA molecules on T cells, B cells and monocytes of 59 healthy subjects and 62 patients with MS who had not received corticosteroids or immunomodulatory therapy in the previous 3 months. RESULTS: The levels of HLA class I and class II molecules expressed on T cells, B cells and monocytes were normal in patients with MS, with the exception of two patients with secondary progressive MS with very low class II expression on B cells. In confirmation of previous studies we also found that the percentage of CD8+ T cells was significantly decreased whereas the percentage of CD4+ T cells and the CD4:CD8 ratio were significantly increased in patients with MS compared to healthy subjects. CONCLUSIONS: The decreased CD8+ T cell response to EBV-infected B cells in MS patients is not due to decreased HLA class I expression on monocytes or B cells. In a small proportion of patients decreased HLA class II expression on B cells might impair the CD8+ T cell response to EBV by reducing CD4+ T cell help.

The role of latitude, ultraviolet radiation exposure and vitamin D in childhood asthma and hayfever: an Australian multicenter study.

Observations of increasing allergy prevalence with decreasing distance from the Equator and positive associations with ambient ultraviolet radiation have contributed to a growing interest in the possible role of vitamin D in the etiology of allergy. The aims of this study were to describe any latitudinal variation in the prevalence of childhood allergy in Australia and to evaluate, in parallel, the individual associations between ultraviolet radiation (UVR)- and vitamin D-related measures and hayfever asthma and both conditions. Participants were population-based controls who took part in a multicenter case-control study, aged 18-61 yr and resident in one of four study regions ranging in latitude from 27°S to 43°S. Data were derived from a self-administered questionnaire, interview and examination by a research officer and biologic sampling. Latitude and longitude coordinates were geocoded from participants' residential locations and climatic data were linked to postcodes of current residence. Stored serum was analyzed for 25-hydroxyvitamin D concentrations and silicone rubber casts of the skin were used as an objective measure of cumulative actinic damage. There was an inverse latitude gradient for asthma (a 9% decrease per increasing degree of latitude); however, this pattern did not persist after adjusting for average daily temperature. There was no association between any of the UVR- or vitamin D-related measures and childhood asthma, but greater time in the sun in winter between the ages 6-15 yr was associated with an increase in the odds of having hayfever [adjusted odds ratios (OR) 1.29; 95% CI 1.01-1.63]. Oral supplementation with cod liver oil in childhood increased the odds of a history of having both asthma and hayfever (2.87; 1.00-8.32). Further investigation of the possible role of early vitamin D supplementation in the development of allergy is warranted. Our results also suggest that solar exposure during childhood may be important in allergic sensitization. Plausible explanations, including biologic mechanisms, exist for both observations.

The essential role of Epstein-Barr virus in the pathogenesis of multiple sclerosis.

There is increasing evidence that infection with the Epstein-Barr virus (EBV) plays a role in the development of multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS. This article provides a four-tier hypothesis proposing (1) EBV infection is essential for the development of MS; (2) EBV causes MS in genetically susceptible individuals by infecting autoreactive B cells, which seed the CNS where they produce pathogenic autoantibodies and provide costimulatory survival signals to autoreactive T cells that would otherwise die in the CNS by apoptosis; (3) the susceptibility to develop MS after EBV infection is dependent on a genetically determined quantitative deficiency of the cytotoxic CD8+ T cells that normally keep EBV infection under tight control; and (4) sunlight and vitamin D protect against MS by increasing the number of CD8+ T cells available to control EBV infection. The hypothesis makes predictions that can be tested, including the prevention and successful treatment of MS by controlling EBV infection.

Associations between silicone skin cast score, cumulative sun exposure, and other factors in the ausimmune study: a multicenter Australian study.

Past sun exposure is linked to a wide range of disease outcomes but is difficult to measure accurately. Silicone skin casts measure skin damage, but some studies show that age rather than sun exposure is the most important determinant of cast score. We examined skin damage scores from silicone casts of the back of the hand in a large adult sample (n = 534) with a broad range of past cumulative UV radiation (UVR) doses. Participants were ages 18 to 61 years and resided in one of four locations down the eastern Australian seaboard, spanning 27-43 degrees S. Data were collected by questionnaire and during a nurse-led interview and examination. Silicone casts were graded from 1 to 6, where higher score represents greater damage. Higher skin damage score was associated with lighter skin pigmentation [adjusted odds ratio (AOR), 4.51; 95% confidence interval (95% CI), 2.33-8.75], fairer natural hair color, particularly red hair (AOR, 11.31; 95% CI, 4.08-31.36), and blue/gray eyes (AOR, 1.72; 95% CI, 1.14-2.59). Higher cumulative UVR dose, particularly before age 18 years, was associated with higher skin damage score (AOR, 2.06; 95% CI, 1.15-2.67 per 1,000 KJ/m(2)), as was number of sunburns, even after adjustment for cumulative UVR dose (AOR, 2.86; 95% CI, 1.50-5.43 for >10 sunburns ever compared with no sunburns ever). Silicone casts of the dorsum of the hand provide a measure of cumulative UVR dose and number of sunburns over the lifetime, which persists after adjustment for chronological age. They can be used as an objective measure of cumulative past sun exposure in epidemiologic studies, but other determinants of skin damage, such as skin pigmentation, should be concurrently evaluated.

Preventing and curing multiple sclerosis by controlling Epstein-Barr virus infection.

It has been noted since 1981 that late primary infection with the Epstein-Barr virus (EBV) in adolescence or young adulthood can account for the epidemiology of multiple sclerosis (MS), including the association with higher socio-economic status, the latitudinal variation in prevalence, the effects of migration on the risk of acquiring MS, and the occurrence of clusters and epidemics. Virtually all (>99%) people with MS are EBV seropositive, suggesting that EBV infection is essential for the development of MS. The EBV-infected autoreactive B-cell hypothesis of autoimmunity published in 2003 proposes that, in genetically susceptible individuals, EBV-infected autoreactive B cells seed the target organ where they produce pathogenic autoantibodies and provide costimulatory survival signals to autoreactive T cells. This hypothesis makes several predictions, some of which have been verified in the case of MS, namely: the presence of EBV-infected B cells in the central nervous system; a beneficial response to B-cell depletion with rituximab; and decreased CD8(+) T-cell immunity to EBV-infected B cells. The hypothesis also predicts that chronic autoimmune diseases will respond to treatment which controls EBV infection. Thus it is proposed that vaccination against EBV will prevent MS, and that effective antiviral drugs will inhibit disease progression in people with MS and potentially be curative.

Study of leukemia inhibitory factor polymorphism within an Australian multiple sclerosis population.

OBJECTIVE: To examine a polymorphism within the 3' untranslated region of the leukemia inhibitory factor gene for an association with multiple sclerosis within an Australian case-control population. METHODS: A test group of 121 unrelated multiple sclerosis patients, of Caucasian origin, and 121 controls, matched for ethnicity, sex and age (+/-5 years) were included in the study. The LIF 3' UTR StuI polymorphism was genotyped by restriction fragment length polymorphism analysis. Statistical analysis of genotype and allele frequencies included Hardy-Weinberg law and conventional contingency table analysis incorporating the standard chi-squared test for independence. RESULTS: Allelic and genotype frequencies did not demonstrate a significant association between the case and control groups for the tested LIF 3' UTR StuI polymorphism. CONCLUSION: The results indicate that the LIF 3' UTR StuI polymorphism is not associated with multiple sclerosis, however we cannot exclude the hypothesis that other polymorphic alleles of LIF could be implicated in MS susceptibility.

Treating autoimmune demyelination by augmenting lymphocyte apoptosis in the central nervous system.

The elimination of autoreactive T cells from the central nervous system (CNS) by apoptosis plays an important role in switching off autoimmune attack. B-cell apoptosis in the CNS probably also has a key role in downregulating autoimmunity. Augmenting lymphocyte apoptosis in the CNS is a potential strategy for treating autoimmune CNS diseases such as multiple sclerosis. These strategies involve modulation of the physiological pro-apoptotic and anti-apoptotic pathways that control lymphocyte fate in the CNS. In the case of T cells, apoptosis can be augmented by enhancing activation-induced T-cell apoptosis through the CD95 (Fas) pathway and by inhibiting costimulation-induced anti-apoptotic pathways mediated through BCL-2 and BCL-X L.