2. Involvement of viruses in the pathogenesis of multiple sclerosis: potential mechanisms
2.1. Molecular mimicry
2.2. Bystander activation and epitope spreading
2.3. Bystander activation by superantigens
2.4. Emerging mechanisms
3. Viral candidates potentially involved in the pathogenesis of MS
3.1.1. EBV: introduction
3.1.2. History of infectious mononucleosis
3.1.3. Seropositivity in MS
3.1.4. EBV presence in the CNS
3.1.5. The functional state of EBV-specific CD8+ T cells in MS patients
3.2. EBV contribution to the pathogenesis of MS: possible mechanisms
3.2.1. MS-specific context: Impaired control over EBV lytic phase antigens by CD8+ T cells
3.2.2. CNS bystander damage
3.2.3. Molecular mimicry, cross-reactivity hypothesis
3.2.4. EBV-infected autoreactive B cells hypothesis
3.2.5. Emerging mechanisms: EBV empowers human B cells for autoimmunity
3.2.6. EBV activates human endogenuous retroviruses HERV-W/MSRV
3.3. Varicella-zoster virus (VZV)
3.4. Human herpesvirus-6 (HHV-6)
3.5. Human endogenous retroviruses (HERV)
3.5.1. HERV-W / MSRV
3.5.3. EBV and HERV in MS: a missing link to the pathogenesis of MS?
3.5.4. HERVs association with MS disease activity, progression and treatment response
3.5.5. HERV-W env: potential therapeutical implications
4. Conclusions and future perspectives
The Role of Viruses in the Pathogenesis of Multiple Sclerosis: an Updated Review
Current data suggest that multiple sclerosis (MS) may be considered as a result of a local inflammatory response in the central nervous system (CNS) initiated by environmental factors in individuals with genetic background predisposing to MS. Data obtained to date by massive genome-wide association studies allow to explain only approximately 30% percent of heritability predisposing to MS, which together with other phenomena speak in favour of the contribution of environmental factors to MS development. Among the most discussed ones potentially trigerring MS are persistent infections which may lead to the activation of autoimmune processes in the CNS. Despite active studies of viral factors in MS etiology, many questions remain, including the contribution of various viral infections or their combinations to the complex pathology of MS, immunological and genetic settings predisposing to the development of MS, etc. This review aims to consider the recent evidence for the involvement of different viruses in the pathogenesis of MS, potential virus-associated mechanisms triggering the disease, and perspectives for MS treatment arising from these findings. In recent years, data obtained by a significant number of independent studies indicate the pathogenetic role of the Epstein-Barr virus (EBV) and human endogenous retroviruses (HERV) in multiple sclerosis, which gives a ground for new therapeutic strategies for this disease.
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) which is one of the most common causes of non-traumatic neurological disability in young adults. MS is characterized by tissue inflammation, demyelination, axonal damage and loss of neurons, resulting in characteristic multifocal lesions on magnetic resonance imaging (MRI) and episodic or progressive neurological disability [1,2]. These lesions typically develop in white matter, where the primary targets are the myelin sheath and myelinating cells, oligodendrocytes; however, grey matter structures (that is, neurons and synapses) of the brain are also affected. A characteristic hallmark leading to MS is increased blood-brain barrier permeability, followed by a pronounced infiltration of immune cells from the periphery into the CNS. Pathological examinations of post-mortem brain samples of MS patients show that the multifocal inflammatory infiltrates in MS brain consist mainly of activated macrophages, T cells, B cells [1,3]. These infiltrated cells are associated with degradation of myelin, axons, oligodendrocytes, and with activation of microglia (resident CNS macrophages) . Within the T cell population, CD8+ T cells are the most abundant, significantly outnumber CD4+ T cells and preferentially show clonal expansion and an activated cytotoxic phenotype in MS lesions . A correlation between the amount of CD8+ T cells and the extent of axonal damage supports the important role of cytotoxic T cells in brain tissue damage via their production of perforin and granzymes . Active MS lesions show a profound heterogeneity, suggesting the heterogeneity of pathogenetic mechanisms and mechanisms of demyelination in different disease subgroups . However, histopathological examinations allowed to distinguish four fundamentally different patterns of demyelination . Numerous studies have established the association between inflammation and neurodegeneration in all MS stages/clinical courses and lesion types, and that inflammation appears to be the driving force for neurodegeneration [8,9].
On a clinical level, in most cases MS is preceded by the clinically isolated syndrome (CIS) as the first single clinical event. MS is classified into four main clinical courses: relapsing-remitting (RRMS), primary progressive (PPMS), secondary progressive (SPMS), and progressive-relapsing (PRMS). Most patients (>80%) initially experience a relapsing-remitting disease course, which is defined by relapses of active disease and phases of remission within which patients recover. In most cases over time RRMS turns into a SPMS form, with continuous disease progression and accumulating disability. In primary progressive MS (PPMS) the disease progresses from onset without any relapses or recovery. Some patients suffer relapses of acute disease activities within a progressive type of clinical disease course (progressive-relapsing MS, PRMS) [10,11].
Studies of brain samples of patients with different types of MS found that pronounced inflammation in the brain is not only present in acute and relapsing multiple sclerosis but also in cases of primary and secondary progressive disease. T- and B-cell infiltrates correlated with the activity of demyelinating lesions, while plasma cell infiltrates were most pronounced in patients with SPMS and PPMS and even persisted . Recent advances in defining more accurately the progressive phenotype of MS have concluded that differences between primary and secondary progressive forms of disease are relatively quantitative rather than qualitative .
The most consistent immunologic finding in patients with MS is the presence of oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF). OCBs arise from the intrathecal synthesis of clonal IgG and are present in more than 95% of patients with MS [13,14]; the pattern of OCBs was found to remain stable over time . Currently, OCBs are one of the most reliable immunological biomarkers with diagnostic and prognostic value in MS, and their detection is still the criterion used to discriminatively diagnose MS in many clinics .
Current data suggest that MS may be considered as a result of a local inflammatory response initiated by environmental factors in individuals with certain immunological settings and genetic background predisposing to MS [17,18]. The time lag of the clinical onset of MS can be explained by that a series of immune-mediated processes in genetically susceptible individuals must reach a “threshold” to begin the disease process . However, the contribution of different factors promoting the onset of immunopathological process in MS still has to be elucidated.
Among the environmental MS risk factors, the most closely investigated to date include 1) latitude, reduced sunlight exposure and Vitamin D deficiency (odds ratio [OR] ≈ 20); 2) prior Epstein-Barr Virus (EBV) infection in early adulthood and adaptive immune response to EBV (OR = 12.5), which will be considered later; 3) smoking (OR = 1.4), and 4) the “hygiene hypothesis” (OR ≈ 3) [reviewed in 20-22]. As the aim of this review is to concentrate on the viral hypothesis of MS, other environmental factors are beyond the scope of this review.
The “hygiene hypothesis” assumes that exposure to several infectious agents early in life is protective against MS, but there is not a specific agent responsible; it comes from the observation that different pathogens may subsequently cause more severe clinical symptoms if primary infection occurs later in life, and might even be responsible for initiating autoimmune disorders. This hypothesis could explain many features of MS epidemiology, such as the greater rates of MS incidence among individuals with higher education and income and therefore with a hygienic upbringing; the trend toward a later age of infection with childhood viruses in MS cases compared to control; the latitude gradient and the apparent protection from MS of individuals born in low-risk areas who migrate to high-risk areas [22,23].
Among genetic factors, Human Leucocyte Antigen (HLA)-genotype carries the strongest genetic burden for MS, although non-HLA genes and gene-gene interactions (epistasis) also play a role. The best documented HLA allele in European populations is DRB1*15:01, which is the strongest risk factor for MS (odds ratio OR ≈ 3.08) . The principal function of major histocompatibility complex (MHC) II class proteins is to present antigens to CD4+ T cells, and these T cells are supposed to have a pathogenic role in MS, although there has been increasing recognition of the importance of CD8+ T cells in the pathogenesis of MS. Interestingly, the MHC class I claster, which regulates cytotoxic T-cell responses, contains polymorphic regions that are associated with the protection against MS . Since different autoimmune diseases share the feature of that risk-conferring genes are encoded within the MHC locus, antigen presentation seems to be of great importance in autoimmune diseases .
Outside the MHC at least 200 variants contribute to MS risk, and follow-up experimental observations have confirmed the role of adaptive and innate immune cells and pathways steering the risk of developing MS. The results also suggest functional responses of brain-resident cells such as microglia and astrocytes affecting susceptibility. However, for most loci the specific DNA variations significant for MS susceptibility and the mechanism linking susceptibility with brain inflammation and autoimmunity remain unknown .
Currently despite massive genome-wide association studies (GWAS), obtained data allow to explain only 20-30% of perceived heritability in MS . The fact that monozygotic tweens are concordant for MS only in approximately 25-30% of cases [28-30] clearly points towards an important role of environmental factors in the development of MS.
Among the most actively discussed factors potentially trigerring MS are persistent infections which may lead to the activation of autoimmune processes in the CNS. In contrast with the suggestion of the autoimmune etiology of MS, the viral hypothesis is more coherent with the pathophysiological process of the disease; the segmentary lesions and the relapse-remission cycles as well as other phenomena which will be discussed later favor the idea of the viral element(s) triggering the immunopathological processes observed in MS . Current data allow to suggest that infectious agents involved in MS pathogenesis are widespread in general population, and that they require specific immunological and genetical context for the development of MS. Despite active studies in this field carried out in recent years, many questions remain, including the contribution of various viral infections or their combinations to the pathology of MS, mechanisms of their involvement in the pathogenesis of the disease, immunological and genetic settings predisposing to the development of MS.
2. Involvement of viruses in the pathogenesis of multiple sclerosis: potential mechanisms
Involvement of viruses in the pathogenesis of MS was suggested a long time ago by numerous observations. Different studies support that viral infections correlate with MS exacerbations [32-39]. Epidemiological studies confirmed the observations that relapses are often preceded by common viral infections such as upper-respiratory infections . Moreover, some studies revealed that exacerbations associated to infectious episodes led to more severe and sustained neurologic deterioration [33,34]. The second point is that CD8+ T cells, which are the main cells involved in viral immunity, rather than CD4+ cells predominate and expand in active MS lesions . The third point is that several viruses are associated with encephalomyelitis, and there are clear examples of demyelination induced by neurotropic viruses in animals [41,42]. The fourth point is that some viruses, including human herpesviruses, are characterized by latency and periodic reactivation, which is a behavior very similar to the pattern of relapsing-remitting MS .
Several mechanisms have been proposed to explain how viruses can trigger autoimmune pathological processes leading to MS, in general they include molecular mimicry and/or bystander activation, which are not mutually exclusive.
2.1. Molecular mimicry
It is known that T-cell receptors (TCRs) recognize short peptide fragments that are generated from foreign or self proteins which undergo an uptake, digestion and protelytic cleavage by antigen-presenting cells (APCs) with a subsequent load onto MHC molecules.
Molecular mimicry or cross-recognition by T-cells occurs when pathogen-specific T-cell receptor (TCR) also recognizes self-antigens due to the high structural similarity between infectious agents and host proteins. Mathematical models indicate that the TCR repertoire is not large enough to provide the specific protection against all possible foreign antigens, and several groups have consistently demonstrated that there can be considerable flexibility in the recognition of peptide-MHC complexes by TCR [37,44]. Polyspecific or so-called degenerate TCR recognition allows to broaden the repertoire of recognized pathogen-derived antigens; at the same time these degenerate T-cell repertoires both escape thymic negative selection and are maintained by peripheral tolerance due to their intermediate affinity recognition of self-peptides presented by self-MHC-molecules. However, certain viral peptides may stimulate autoreactive T cells under special circumstances, e.g., strong innate immune activation [44-47].
According to the recent data, cross-reactivities do not necessarily require nearly identical amino acid sequences, they may also occur when some important motifs are conservative which provides that the overall structures of TCR–peptide–MHC interaction are similar . Moreover, the binding groove of MHC class I molecules limit the strength of the presented peptides to 8-10 amino acid groups, even though examples of longer peptides presented on MHC class I molecules exist. Taken together, these phenomena increase the probability that T cells specific for certain viral antigens may also be cross-reactive for self-antigens.
Research on possible infectious triggers of MS has shown that T cell clones specific for an immunodominant epitope of MBP (myelin basic protein), MBP85-99, also recognize different viral antigens, such as EBV, human herpesvirus-6 (HHV-6), influenza A virus, herpes simplex virus (HSV) . Different studies reported about homologous sequences or structural mimicry of EBV and human herpesvirus-6 (HHV-6) with MBP [48-50].
Libbey and Fujinami have suggested that molecular mimicry of different viral infections to different CNS antigens and multiple mimicry pathways could explain the inability to isolate single causative agent in MS and could explain the phenotypic heterogeneity of the disease, respectively . However, it has become clear that structural similarities between viral and self proteins are usually not sufficient to lead to pathological consequences, and in healthy immune system multiple mechanisms work to prevent pathologic consequences of cross-recognition. Autoreactive T cells with high affinity for self-MHC/self-antigen complexes are usually eliminated by clonal deletion in thymus. If autoreactive T cells escape deletion due to thymic lack of expression of some autoantigens, or due to the low affinity of their TCRs, then peripheral tolerance mechanisms such as T-cell anergy, activation-induced cell death and regulatory T cells provide further control over autoreactive T cells. Despite that our current understanding of the context triggering the initial pathological autoimmune response in humans is incomplete, most likely the combination of factors contributes. These probably include the susceptibility-conferring genetical background as well as an infectious context and strong activation of both innate and adaptive immune systems. This activated state leads to a shift of the immune response towards a proinflammatory T cell phenotype (T helper 1, Th1) and may be induced by such conditions as high antigen dose and the presence of proinflammatory cytokines; viral tropism to the target tissue; local tissue destruction and/or immune activation in the target tissue .
2.2. Bystander activation and epitope spreading
Bystander activation represents another possible mechanism by which peripheral infection could promote MS onset or exacerbation. Bystander T cells may be defined as T cells that migrate to a site of inflammation, but are not specific for appropriate viral antigens or for a self-proteins at the site of inflammation.
According to this theory, the Th1-driven environment promoted by systemic viral infection could contribute to the activation of bystander autoreactive T and B cells via proinflammatory cytokines.
Although microbial infections are able to induce proinflammatory cytokine secretion pattern, cytokines alone are unlikely able to drive functional activation or expansion of T cells in the absence of cognate antigen, which have been demonstrated on animal models . Interestingly, the data of Correale et al. demonstrated that in MS patients in the presence of an infectious agent, myelin-specific T cell lines (T cells specific to peptides of MBP and myelin oligodendrocyte glycoprotein (MOG)) were activated by far lower peptide concentrations than those required for successful activation by the cognate peptide alone .
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Fig.1. Potential mechanisms underlying infection-induced autoimmunity (explanations are given in the text)
(Author’s own work)
Alternative scenario of bystander activation occurs when an over-aberrant antiviral immune response result in the release of self-antigens not usually exposed to the immune system. These self-antigens may be captured and processed by APCs such as dendritic cells and then presented to autoreactive T cells. T cells activated in a bystander manner are thought to have low affinity for self-antigen, so their subsequent activation requires some form of immunological adjuvant, with viral infection as a primary candidate. Enhanced processing and presentation of self-antigens may result in the spread of the immune response towards different self-antigens, a process called epitope spreading [47,52,54].
In addition, virus-sprecific T cells also might initiate bystander activation of autoimmune response: they may migrate to areas of viral infection including CNS, where they recognize infected cells and release cytotoxic granules resulting in the killing of infected cells. Under these circumstances the dying cells, the CD8+T cells and inflammatory cells (macrophages and microglia if we consider the CNS damage) within the inflammatory focus release cytokines such as tumor necrosis factor TNF-β, lymphotoxin, and nitric oxide (NO), which can lead to bystander killing of the uninfected neighboring cells. This results in additional immunopathology at sites of infection [52,54].
2.3. Bystander activation by superantigens
Bystander activation may also be achieved by superantigens, which are produced by viruses and bacteria, don’t need to be processed by APCs as conventional antigens and activate T- and B-cells regardless of the specifity of their antigen receptor. They bind to conserved regions of MHC II outside the classical peptide-binding groove and connect them with certain variable Vβ regions of the T-cell receptor (TCR). Therefore, superantigens can stimulate many subsets of T cells expressing the same Vβ genes, followed by massive cytokine secretion . The polyclonal population of T cells activated by superantigens may contain a T cells specific for self-antigen . Staphylococcal, mycoplasma, endogenous retrovirus-derived, and enteric microbiota-generated superantigens were suggested to be involved in the disease exacerbations in animal models of MS .
2.4. Emerging mechanisms
Recently, another interesting mechanism of broken MBP-specific CD8+ T cell tolerance was described, according to which CD8+ T cells expressing a dual TCR receptor capable of recognizing both MBP and viral antigens may be activated by a virus that doesn’t express myelin cross-reactive epitopes and doesn’t depend on bystander activation . As the frequency of T cells co-expressing a myelin-specific and a virus-specific TCR in the peripheral T cell repertoire should be low, and will likely vary among individuals, this mechanism may represent one of the ways by which a common viral infection can trigger autoimmunity in a small subset of genetically predisposed individuals .
Furthermore, Epstein-Barr virus (EBV) is known to immortalize B cells and facilitates their differentiation into long-lived memory B cells. These mechanisms could promote the survival of autoreactive B cells infected with EBV with their possible role as antigen-presenting cells that can present autoantigens and contribute to the autoimmune process in the CNS . Recent experimental study  support that EBV may change processing of myelin antigens and facilitate cross-presentation of disease-relevant epitopes to CD8+ T cells by infected B cells. However, the question of whether EBV-infected B-cells in the MS brain have autoreactive properties remains to be elucidated.
The proposed mechanisms according to which infectious agents potentially initiate and support pathological processes underlying MS are dynamic, not mutually exclusive and might occur simultaneously or sequentially. A simple “one mechanism – one disease” paradigm might not correspond to complex and heterogeneous diseases such as MS . Fujinami et al. proposed the “fertile field” hypothesis, according to which infections can induce a heightened immunologic state, which in the presence of additional antigens (viral, environmental, or self) can lead to the expansion of autoreactive T cells. Later, in the presence of additional antigens (viral, environmental, or self), this expanded pool of autoreactive T cells may develop into autoaggressive cells. This concept incorporates the idea of a threshold for autoimmunity, which is partly due to an individual’s genetics, and is regulated by the quality and magnitude of infections and the host immune responses to those infections .
3. Viral candidates potentially involved in the pathogenesis of MS
Numerable studies showed the association of MS with different infectious agents, and no single infectious agent allows to completely explain the complex pathogenesis of the disease. The CSF of MS patients is characterized by polyspecific antiviral humoral immune response, with the intratecal synthesis of IgG directed against a variety of viruses including measles, rubella, varicella zoster virus, and, to a significantly smaller proportion, against HHV-6 and EBV [60-63]. The presence of antibodies against measles, rubella, and varicella zoster virus (MRZ reaction, MRZR) was observed in the CSF of 80-100% of MS patients but was less common in acute inflammatory diseases of the CNS while being observed in only 3% of the controls [62,64]. Although antibodies to these viruses are the most frequent constituents observed in the CSF of MS patients and show high diagnostic value for predicting conversion of clinically isolated syndrome in MS [62,65], there is no evidence that they contribute to the initiation and aggravation of CNS damage in MS.
Given the association of viral infection with MS relapses, it is reasonable to hypothesize that the relapsing-remitting pattern of the initial stages of disease could perhaps be explained by the latency and reactivation of various chronic viral infections widespread in a general population , among which the members of Herpesviridae family seem to be attractive culprits because of their neurotropism, ubiquitous nature, and tendency to produce latent, recurrent infections .
Herpesviruses represent a group of double-stranded DNA viruses, including eight species that infect humans. After primary infection which typically occur in a childhood, all herpesviruses remain latent within specific host cells and may subsequently reactivate. Reactivation is not necessarily associated with clinical symptoms, as reflected in the asymptomatic shedding of virions from oral mucosa. There are eight human herpesviruses (HHVs): herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV or HHV-4), cytomegalovirus (CMV or HHV-5), HHV-6, HHV-7 and HHV-8 (Kaposi sarcoma (KS) herpesvirus). At least five species of the Herpesviridae - HSV-1 and HSV-2, VZV, EBV and CMV – are extremely widespread in the general population. Most of the HHVs are neurotropic and a common cause for serious acute and chronic neurological disease of the CNS; this can be monophasic, recurrent or chronic [67-69].
Herpesviruses are divided into three groups. The alpha subfamily (VZV and HSV-1 and -2) primarily target neurons for long-term habitation, but also replicate in mucoepithelial cells, which is essential for viral shedding through skin or mucosa. B eta and gamma subfamilies (to which belong CMV, HHV-6, HHV-7, and EBV, HHV-8, respectively) have a tropism for various subsets of leukocytes, but most of them can also infect epithelial cells . The delicate balance between latency and reactivation of herpesviruses is designed by evolution. In case of the normal interaction of the healthy host with the virus, the process is tuned to a long-term relationship that does not cause excessive harm. However, if this balance is disturbed by environmental factors, or if the host is immunocompromised, the virus may inadvertently cause disease. .
Among human herpesviruses, infection with EBV, HHV-6 and VZV has particularly been associated with MS, although the data concerning the involvement of the latter two viruses appeared to be controversial and require additional studies.
EBV is almost ubiquitous B-lymphotropic human DNA γ-herpesvirus which infects more than 90% of world population. Latently infected B cells can express three different transcriptional programs of EBV depending on the differentiation state of the infected B cell.
During primary infection, EBV, transmitted via saliva, infects naïve B cells in the tonsils. After entering naïve B cell, EBV immediately activates expression of two anti-apoptotic proteins of vBcl-2 family, BHRF1 and BALF1, which are essential for the survival of infected B cells , and activates so-called ‘growth’ or ‘latency III’ transcriptional program, which drives the infected B cell to become proliferating lymphoblast (fig. 2). Latency III or ‘growth’ programme activates expression of nine latent EBV proteins including six nuclear proteins (Epstein-Barr virus nuclear antigens EBNAs- 1, -2, -3a, -3b, -3c, and LP) and three membrane proteins (latent membrane proteins LMP1, LMP2a, LMP2b); in addition to these proteins, EBV-infected lymphoblasts express two small non-coding RNA, termed EBER1 and EBER2, and approximately 40 miRNA. These EBV-infected B cells proliferate, and after the initial clonal expansion, some of them enter a tonsillar germinal center. To successfully drive the infected B cell through germinal center reactions, at this stage EBV restricts its expression pattern to EBNA1, LMP1 and LMP2 (latency II or ‘default’ programme). The clonal expansion in the germinal center allows to increase the number of EBV-infected B cells. These B cells then differentiate into latently infected memory B cells, exit from the germinal centre and circulate in the blood; they express no viral proteins except during cell mitosis, when they express only EBNA1 (latency I). As latently infected memory B cells are resting, long lived and have the phenotype of classical memory B cells, EBV can persist in these cells for a long time without expression of any of the known latent viral proteins. This strategy also allows virus to escape the recognition by EBV-specific T cells. If latently infected memory B cells become reactivated and differentiate into plasma cells, EBV switches to lytic life cycle, producing many new virus particles, some of which might infect new naïve B cells. Thus EBV exploits the normal B cell differentiation pathways to establish a life-long persistence in the B-cell compartment [71-75].
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Fig.2. EBV lifecycle (explanations are given in the text) (Author’s own work)
Special attention should be paid to the peculiarities of EBV-infected B cells switching to the ‘default program’ during germinal centre reactions.
It is known that in classical B cell differentiation, naive B lymphocytes activated by antigen via B cell receptor (BCR) and then by T cells through CD40L-CD40 interactions, proliferate and pass through reactions in the germinal center. The BCR produces two types of signals: one (tonic) is required for the survival of resting B cells, while the other (activating) leads to cellular activation, proliferation and ultimately differentiation into immunoglobulin-secreting plasma cells. It has been shown that latent membrane proteins LMP2A and LMP1, expressed on B cells by EBV during latency III and latency II stages, mimic BCR and CD40, respectively, providing constitutive, ligand-independent signals and thus allowing these B cells to pass through the germinal center reactions independently of T cells [71,76]. Studies in vitro demonstrated that LMP2A is able mimic and replace BCR in a BCR-mediated signaling, thereby maintaining apoptosis-resistant B lymphocytes in an active, proliferating state. In transgenic mice, LMP1 can also act as an active CD40 receptor that completely replaces CD40 in a CD40-mediated signaling, resulting in the normal development of B cells, their activation and the ability to switch classes of immunoglobulins, germinal center formation and somatic hypermutation .
Evidence for the role of EBV in the pathogenesis of MS
3.1.2.History of infectious mononucleosis
EBV transmission typically occurs via infected saliva, and in almost all EBV-seropositive individuals the EBV virions shed into saliva, which allows the virus to infect EBV-naïve individuals. The majority of EBV infections are asymptomatic and occur in early childhood, whereas up to 50% of primary EBV infection in adolescents and adults cause infectious mononucleosis, with varying degrees of clinical severity .
A few decades ago it has been noted that primary EBV infection at an earlier age typically occurs in areas where MS is less common, while EBV infection at a later age is observed in areas with high prevalence of MS [77,78]. Indeed, primary infection with EBV at a later age can explain epidemiological features of MS, such as the association with higher socio-economic status; the latitudinal variation MS prevalence increasing with distance from the equator; the occurrence of clusters with higher MS incidence rates; and the effects of migration on the risk of acquiring MS .
Evidence for a potential role of EBV in the development of MS arises from resent meta-analyses of numerable studies which confirmed the association between infectious mononucleosis (IM) and MS [79-81]. The study of Goldacre et al. showed the 4-fold increase in the risk of the development of MS after clinically confirmed IM . The mean time interval between IM and MS onset in this study was 14 years. In the large-scale study of Ramagopalan et al. (14,000 MS cases and 7000 controls) a positive correlation of MS cases with history of IM was found, while no such association was observed for history of measles, mumps, rubella, hepatitis B .
3.1.3.Seropositivity in MS
Seroepidemiological studies of recent 3 decades have shown that the levels of EBV-serpositivity in MS patients are higher than that in control group (99,5% compared to 94%); but much more convincing evidence has been obtained in groups of children with MS – 83-99% compared to 42-72% in age-matched controls . It is notable that such high levels of seropositivity were observed for EBV but not for other viruses [85,86]. Recent metaanalysis of 25 studies with the use of two independent methods for determination of EBV seropositivity allowed to conclude that EBV infection is present in 100% of MS patients . Another recent metaanalysis of studies with two independent EBV detection methods (ELISA and immunoblotting) in patients from different countries with clinically isolated syndrome (CIS) who converted to clinically defined MS has established a truly negative seropositivity only in one patient from 1047 (<0,01%) , therefore supporting that EBV is an essential prerequisite for the development of MS and that MS cases in EBV-seronegative patients occur extremely rare.
The longitudinal study of Levin et al. investigated the presence of antibodies to EBV before MS onset. It has been found that serum antibody titers to EBNA complex were similar between controls and individuals who developed MS before the age of 20, but 2- to 3-fold higher in MS cases of age 25 years and older . These results suggest an age-dependent relationship between EBV infection and development of MS.
A marked increase in serum antibody titers (IgG) against EBV antigens (mainly EBNA-1 or EBNA-complex) several years before the onset of clinical manifestations of MS suggests the involvement of EBV in early stages of the pathogenesis of MS [90-92]. This early increase in anti-EBNA antibodies may be due to the spread of EBV into the CNS, which probably occurs several years before the clinical symptoms of MS. The study of Lünemann J.D. et al. found that, compared with the control group, patients with CIS had stronger humoral and cellular immune responses to EBNA1, but not to other EBV antigens . It is noteworthy that among the analysed infections EBNA-1 was the only antigen the levels of antibodies to which correlated with the number of active demyelinated lesions on MRI (Т2-lesions), and were found to correlate with the future disease progression measured by EDSS (Expanded Disability Status Scale) and MRI . Obtained data point to selective increase in immune responses to EBNA1 in CIS patients and suggest that anti-EBNA1 IgG titers may be used as a prognostic marker of CIS to MS conversion and disease progression. A pronounced positive correlation between elevated levels of anti-EBNA1 IgG and MS activity observed on MRI and measured by the number of new active lesions has been confirmed by other studies [94-96]. Given that EBNA1 is the only EBV protein expressed by homeostatically proliferating memory B cells, the predominance of IgG-response directed against EBNA1 observed in MS patients may reflect a high frequency of latently infected B cells but not the specific pathogenic role of these antibodies .
It is important to distinguish the role of EBV in MS from that of other infectious agents. It has been established that MS exacerbations occur three times more likely at the time of acute systemic infections with a wide variety of viruses and bacteria. EBV differs from other infectious agents in many ways. First, the initial onset of MS occurs years after primary EBV infection, whereas other infections precede MS exacerbations by only one or two weeks. Second, EBV persists for the lifetime of the host, in contrary to the infections preceding MS exacerbations. Third, EBV is the only agent infecting virtually all MS patients. Fourth, EBV is the only virus that infects and modulates B cells involved in the disease, which will be discussed later. These features of EBV infection suggest the essential role of this virus in the development of MS .
3.1.4.EBV presence in the CNS
According to different studies, B-lymphocytes and plasma cells are an integral part of inflammatory infiltrates in white matter and meninges in MS [97-99], and the most pronounced accumulation of B cells in MS brains referred to as “ectopic follicles” or “follicle-like structures” has been observed in patients with a progressive type of disease with severe inflammatory and neurodegenerative processes [100-105]. B cells have also been found to be significantly increased in MS cerebrospinal fluid .
The presence of oligoclonal bands in cerebrospinal fluid, which result from local antibody production, observed in the vast majority of patients already at MS onset, indicate that abnormal B cell activation might be an early event in this disease . Indirect evidence for transformation of B cells in the CNS of MS patients comes from the observations that the pattern of OCBs in cerebrospinal fluid of MS patients remain stable over time, and further examinations found no MS-specific pattern in specifities of these antibodies. The possible explanation for these phenomena may be in that such specifities are random as it observed in EBV-transformed and immortalized B cells .
Studies on B cell-depleting therapies, which show promising results, has led to the reassessment of the role of B cells in the pathogenesis of MS. In particular, anti-CD20 therapy (rituximab, ocrelizumab) was found to significantly limit new focal inflammatory brain lesions and disease relapses in relapsing-remitting MS, and may limit worsening of disability in cases of primary-progressive MS . CD20 depletion targets only B cells, and does not immediately affect plasma cells or secreted antibodies levels . The results of clinical trials of these drugs indicate abnormal pro-inflammatory properties of B-cells in MS, as well as their ability to influence the activity of the disease regardless of the production of antibodies. Different studies speak in favor of this suggestion. B cells can function as antigen-presenting cells able to initiate T-cell-specific responses; moreover, B cells are highly selective APCs, as they recognize their antigen via their BCR. BCR-captured antigen is subsequently internalized, processed and presented to T cells. Several studies have demonstarted that B cells from MS patients show increased levels of expression of costimulatory molecules CD80 and CD86 . Another study has also found that peripheral B cells in MS patients show a shift towards antigen-experienced memory B cells (expressing a memory B-cell marker CD27+) with increased production of proinflammatory cytokines, contributing to the dysregulated inflammatory milieu in MS . In the cerebrospinal fluid of MS patients, CD27+ memory B cells were also found to have an upregulation of costimulatory molecules, which suggests an active interaction between B and T cells . In support of this, some studies have shown that rituximab therapy in MS patients reduces CSF counts of both B cells and T cells (95% and 50% mean reductions, respectively) .
The study of Harp et al. has suggested that a proportion of peripheral memory B cells in patients with RRMS can directly present myelin antigens (MOG and MBP), thus eliciting CD4+ T-cell activation and proliferation, with the potential to promote pro-inflammatory differentiation of responding T cells .
The next point regarding the pathogenetic role of B cells in MS is associated with the fact that CD27+ memory B cells can also serve as natural reservoirs of latent EBV infection, and both B cells and EBV seem to be necessary factors in MS . Direct confirmation of this was provided by the study performed on postmortem brain samples of patients with MS, which demonstrated that most B cells in MS lesions, meninges and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent EBV proteins LMP1 and LMP2, that provide survival and maturation signals to B cells .
In the last decade, convincing data have been obtained on the probable relationship between acute inflammation, EBV reactivation and cytotoxicity toward EBV-infected B cells in the central nervous system in patients with MS.
In the study of Serafini et al. it has been found that a significant portion of B cells and plasma cells in postmortem brain sections of 21 out of 22 patients with MS were infected with EBV, while no infected B cells were detected in the brain sections of patients with other inflammatory diseases of the CNS . The main sites of EBV persistence in brain samples of patients with secondary-progressive MS were meningeal structures resembling B cell lymphoid follicles with germinal centers. Expression of EBV latent proteins EBNA2 and LMP1 was regularly observed in MS brains, while viral reactivation was restricted to ectopic B cell follicles and acute lesions, where a significant number of B cells expressing the EBV lytic cycle-associated protein, BFRF1, was detected. What is of particular importance in this study is that CD8+ T cells infiltrated all the sites in the CNS where infected B cells were located, including ectopic B-cell follicles in cases with progressive clinical course, and that the number of CD8+ T cells strikingly correlated with the number of EBV-infected B cells. Prominent accumulation of CD8+ T cells was also observed in active lesions of the two acute MS cases, matching the distribution of EBV-infected B cells . These data give the evidence that persistence and reactivation of EBV and cytotoxicity directed toward EBV-infected B cells in the central nervous system in MS patients play an important role in immunopathology of the disease.
Serafini et al. identified EBV-infected B cells using EBER-ISH (in-situ hybridization (ISH) targeting EBV-encoded small RNA (EBER)) and by immunohistochemistry with antibodies specific for different EBV proteins. EBER-ISH is recognized as the gold standard for detection of EBV-infected B cells in histological material because EBER, which are non-coding RNA transcripts, are abundantly expressed by B cells during all phases of EBV latent infection .
At the same time, several research groups couldn’t detect EBV in the central nervous system of patients with MS [115-117], which has been the subject of an active discussion in recent years . Possible explanations for such discrepancy in results are methodological differences that may lead to differences in the sensitivity of EBV detection. Features of tissue processing and fixation can significantly affect the safety of EBV-encoded small RNAs (EBER) – in particular, EBER RNA is degraded by long tissue fixation times in formalin . Another factors that also have a great influence on the results are the number and selection of brain tissue specimens for analysis, the degree of CNS infiltration by B cells, the sensitivities and specificities of the methods used, as well as interpretation of the studies. In addition, there is a wide spectrum of pathological features of multiple sclerosis lesions,depending upon the type and severity of the disease as well as the stage of the lesions [118-120].
Results confirming the findings of Serafini B. et al. were obtained in studies using similar research methods and methodological nuances in brain samples processing [120-122]. A recent independent study, which also used PCR, EBER-ISH and immunohistochemistry, established the presence of EBV in brain samples in 90% (91 of 100) of MS cases analyzed . Data obtained with the immunohistochemical method using double staining indicate that not only B cells, but also astrocytes and microglia in brain sections of patients with MS (10-15% of the total the number of cells detected as EBER+ in brain samples) are infected with EBV .
Another line of evidence about the presence of EBV in the central nervous system of patients with MS comes from other studies. Effector cytotoxic CD8+CD57+ T cells, which seem to have a central role in eliminating EBV-infected B cells by release of IFN-γ, were found in meningeal inflammatory infiltrates from postmortem brain samples of patients with secondary-progressive MS . This not only confirms the presence of EBV in the CNS, but also indicates the association of this T cells subset with the pathological process in MS. Interestingly, a high frequency of myelin-specific CD8+CD28-CD57+ T cells was discovered in MS patients suggesting that myelin-reactive CD8+ T cells are chronically stimulated in MS patients . In another study, effector memory CD8+ T cells isolated from white matter lesions in brain samples of patients with MS were found to be highly reactive towards autologous EBV-infected B cells . Recent study with the high-throughput sequencing of the TCR-β gene repertoires revealed enrichment of TCR-β sequences of EBV-reactive CD8+ T cells in the cerebrospinal fluid of patients with MS, which was not observed in the control group .
Further indirect evidence for the presence of EBV in the CNS of MS patients comes from the finding that EBV-specific (but not cytomegalovirus, CMV-specific) cytotoxic CD8+ T cells are enriched in the CSF in patients with early MS, but not in patients with other inflammatory neurological diseases ; these data speak in favor of the involvement of EBV-directed CD8+ T cell response in the CNS on early stages of MS. Furthermore, the increase in CD8+ T cells specific to lytic EBV antigens observed in cerebrospinal fluid and peripheral blood of MS patients in active phase of disease suggests the close association between EBV replication in the CNS and inflammatory disease activity [128,129]. The evidence for an active EBV replication in MS brain lesions obtained by several studies [84,121,128,130] allows to suppose the impaired control over EBV by CD8+ T cells in MS.
Therefore, available data indicate the association between EBV reactivation in the CNS of MS patients and cytotoxic CD8+ T cell response directed against EBV. However, due to discrepancies concerning the presence of EBV in brain samples of MS patients, the prevalence of active EBV infection in MS brain still has to be verified in the framework of additional independent studies.
3.1.5.The functional state of EBV-specific CD8+ T cells in MS patients
In healthy individuals, EBV infection is kept under control by humoral and cellular mechanisms - infectious virions are destroyed by neutralizing antibodies, while proliferating and lytically infected B cells are the targets of EBV-specific CD8+ T cells [131,132]. Although virus-specific CD4+ T cells contribute in efficient immune control over EBV, CD8+ T cells are known to play a crucial role in controlling both latent and lytic EBV infection [131-134]. During primary and persistent EBV infection, the frequency of EBV-specific T cells in healthy individuals was shown to be much higher in CD8+ T cell population than in CD4+ T cell subset [133,136]. Therefore, considering the data supporting the involvement of EBV in MS pathogenesis, the question rises about the amount and functionality of EBV-specific CD8+ T cells in MS patients.
Studying EBV-specific cellular immune response relies on two different approaches: one with the use of specific EBV peptides or multimers, while the other employs autologous EBV-infected B cells for measuring CD8+ T cells responses.
Studies using peptides appeared to be inconsistent, with some finding increased [128,136,137], normal [138,139], or decreased  CD8+ T cell responses to some EBV epitopes in patients with MS compared to healthy controls.
Pender et al. using autologous EBV-infected lymphoblastoid cell lines (LCL) showed that MS patients have a decreased frequency of LCL-specific CD8+ T cells compared to healthy subjects . The study of Lindsey et al. found no significant differences in number and cytotoxicity of these cells between MS patients and healthy controls; however, there was a trend towards decreased proliferative responses of LCL-specific CD8+ T cells in MS patients, indicating an aberrant CD8+ T cell control of EBV-infected B cells . Another study showed that CD8+ T-cell cytotoxic responses to latent EBV proteins were higher in MS patients than in controls .
These discrepancies between the results may be due to different reasons . First, the responses measured to selected EBV peptides or multimers represent only a small proportion of the aggregate CD8+ T-cell response to EBV-infected B cells, while the use of autologous LCL express not only latent, but also the lytic EBV proteins. Second, the use of peptides bypasses the normal physiological process of antigen-processing; thus, exogenously added synthetic EBV peptide may stimulate a strong production of IFN-γ by CD8+ T cells, whereas studies using autologous LCL may represent mechanisms closer to natural antigen processing. Third, the stage of the disease at which blood is collected or the way the methods were applied may also lead to inconsistent results .
A recent comprehensive study of Pender et al. also found that CD8+T cells directed against EBV latent antigens were increased, but had reduced cytokine polyfunctionality, indicating T-cell exhaustion in MS patients . Of particular importance is also the finding that at all stages of MS, except during clinical attacks, the CD8+ T cell response was significantly shifted from lytic to latent EBV antigens; the median lytic/latent ratio in CD8+ T cell population was 6-fold lower in patients with MS compared to healthy subjects. During MS attacks, the EBV-specific CD4+ and CD8+T-cell populations expanded, with increased functionality of latent-specific CD8+T cells. In contrast, CD8+T-cell response to EBV lytic antigens was found to be decreased at the onset of MS and at all subsequent disease stages. With increasing disease duration, EBV-specific CD4+and CD8+T cells progressively declined, consistent with T-cell exhaustion . A progressive decline in frequency of EBV-specific effector CD8+ T cells, which correlated with the duration of MS, was also confirmed by the study of Jilek S. et al. , which has shown that the frequency of EBV-specific cytotoxic CD8+ T cells in blood of patients with CIS was significantly higher than that found in patients with RRMS, PPMS, SPMS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and the assays, the more intense was the EBV-specific CD8+ T-cell response .
Of particular importance is the finding that CD8+CD57+ T cells which seem to have a central role in eliminating EBV-infected B cells express high levels of Programmed Death 1 (PD-1) in blood of RRMS patients in remission phase compared to patients with active MS or healthy donors . PD-1 is an immune checkpoint that regulates activation, proliferation and cytokine release from CD8+ T cells and promotes self-tolerance by suppressing T cells activity; on the other hand, high levels of PD-1 expression are associated with an anergic exhaustion-like phenotype which was described recently in virus-specific CD8+ T cells in chronic viral infections such as HIV, hepatitis B and hepatitis C . The study of Cencioni M. et al. showed that increased expression of PD-1 by CD8+CD57+ T cells of patients with MS correlated with a reduction in release of IFN-γ, perforin, and granzyme B towards EBV-infected B cells. Detection of a higher frequency of PD-1 expressing CD8+CD57+ T cells in the blood of RRMS patients during remission, and concomitantly suppressed degranulation, suggests that the inability of cytotoxic T cells to control EBV replication during inactive MS could set the stage for EBV reactivation in the CNS and disease progression .
In healthy EBV carriers, despite that EBV periodically reactivates with shedding of virions into saliva, the EBV is kept under control by lytic-specific CD8+ T cells. The crucial role of controlling the lytic phase of infection is supported by several studies, including the study of Hopwood et al. , who concluded that in healthy individuals viral loads are maintained within normal limits by cytotoxic CD8+ T cells which target lytic rather than latent EBV proteins . Based on the foregoing, M. Pender et al. proposed a hypothesis according to which on inintial stages of MS impaired CD8+ T-cell control over EBV may lead to the uncontrolled increase in number of EBV-infected B cells, including the unrestrained proliferation of EBV-infected germinal centre B cells, which in turn will increase the infected memory B-cell population and thus the number of latently infected B cells avaible to reactivate the EBV into the lytic stage; this leads to the further aggravation of exhaustion of the latent EBV- specific CD8+ T cells. This vicious cycle might underlie the accumulation of EBV-infected B cells in the CNS and subsequent development of MS .
Based on the above, a 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 CD8+ T cells targeting EBNA, LMP1, and LMP2A.Clinical assessments at the venesection visits and during 27 weeks after the first T cell infusion showed there were no serious adverse events and that therapy was well tolerated. Seven patients showed improvement, with 6 experiencing both symptomatic and objective neurological improvement, together with a reduction in fatigue, better quality of life, and, in 3 patients, reduced intrathecal IgG production. It is worth noting that clinical improvements occurred in patients who had experienced progressive neurological deterioration for a mean of 10.1 ± 6.7 years prior to T cell therapy and did not represent resolution of acute MS relapses . Such promising results add to the mounting evidence for a pathogenic role of EBV infection in MS and set the stage for further clinical trials with autologous EBV-targeted T cell therapy in progressive forms of MS. Further clinical trials are required to determine the efficacy and safety of EBV-specific T cell therapy in MS and its appropriate doses for positive therapeutical effect without side effects. Due to the potential risk of aggravating CNS inflammation, this therapy should probably not be tried yet in patients with relapsing-remitting MS, for which a number of disease-modifying therapies are already available .
3.2. EBV contribution to the pathogenesis of MS: possible mechanisms
3.2.1. MS-specific context: impaired control over EBV lytic phase antigens by CD8+ T cells
Serafini B. et al. have shown that the patterns of expression of EBV proteins in analyzed brain samples of MS patients in most cases indicated "latent phase III" (EBNA2, LMP1) and "latent phase II" (LMP1) of latent EBV infection . Under normal control over EBV, cells expressing LMP1 and EBNA2 are ususally absent in the blood, thus these data indicate complete disruption of EBV regulation, for example due to the inability of CD8+ T cells to eliminate latently infected B cells in the CNS [84,142,148], which was suggested by several studies [84; 128]. Results of different studies support this suggestion, demonstrating that in MS patients subpopulations of EBV-specific CD8+ T cells show signs of depletion [124,141,142,144,149], which allowed M. Pender to suggest that on inintial stages of MS impaired CD8+ T-cell control over EBV leads to the uncontrolled increase in number of EBV-infected B cells and thus the further aggravation of exhaustion of the EBV-specific CD8+ T cells. This vicious cycle might allow EBV-infected B cells to accumulate in the CNS and lead to the subsequent development of MS .
3.2.2.CNS bystander damage
The EBV bystander damage hypothesis complements the previous hypothesis and proposes that immune attack on the CNS observed in MS is primarily directed against EBV antigens, especially lytic antigens, leading to the bystander damage of the CNS [84,128].
The aforementioned data support the link between acute inflammation, EBV reactivation and CD8+ cytotoxicity directed against EBV in the central nervous system of patients with MS. The question remains why EBV-specific CD8+ T cells fail to eliminate EBV-infected B cells in brain on initial stages of MS despite being powerful enough to cause demyelination – is it due to that EBV-infected B cells outnumber the EBV-specific CD8+ T cells in the CNS, is it due to the exhaustion of EBV-specific CD8+ T cells, or another parallel mechanisms involved in MS from a certain point make the pathogenetic processes uncontrolled for immune regulation - for example, the release of myelin antigens with subsequent activation of naïve myelin-reactive T cells in proinflammatory context.
In the framework of this hypothesis, CNS antigens released after a cytotoxic response directed against EBV and causing bystander neuronal damage may lead to the secondary autoimmune responses. Due to the lack of unequivocal evidence for the autoimmune nature of MS [reviewed in 150], the contribution of autoimmune processes in the pathogenesis of MS and settings that trigger their activation, as well as the timing of their inclusion in the process, remain to be elucidated.
Since EBV is very effective at evading CD8+ T cells while latent, if the EBV-infected memory B cell concentration in the CNS is similar to that found in peripheral blood (0.001% of memory B cells infected), such a low load should not elicit an immune response. This may partly explain the rare occurrence of MS (∼0.1% of the population), despite the fact that most of the world’s population is infected with EBV (60-90% of the population, depending on age and environment). However, were EBV-infected memory B cells in the CNS to reactivate, expand and differentiate into plasma cells, viral synthesis would occur, promoting a strong cytotoxic CD8+ T cell response. Theoreically, even a single acute EBV reactivation event in the CNS could lead to a loss of immune tolerance to CNS antigens, though such a hit-and-run mechanism would be difficult to prove .
3.2.3.Molecular mimicry, cross-reactivity hypothesis
According to the hypothesis of molecular mimicry, some T cells specific for EBV antigens (such as EBNA-1) are structurally similar to CNS antigens like myelin basic protein (MBP), and TCR may be able to recognize more than one peptide, which results in the recognition of autoantigens [37,48,49]. In healthy immune system multiple mechanisms work to prevent pathologic consequences of cross-recognition , but under special circumstances, such as strong innate immune activation and proinflammatory context, high viral antigens load, local tissue destruction, certain viral peptides may stimulate autoreactive T cells [37,44-47].
Several studies have demonstrated the presence of T cells in MS patients which recognize both EBV and myelin epitopes [49,138,152]. EBV-specific T cells cross-reacting with MBP were found in the CSF of a patient with MS . It has also been reported that EBNA1-specific CD4+ T cells from some MS patients recognized myelin antigens more frequently than other autoantigens not associated with MS, and produced IFN-γ upon stimulation, which allows to suggest the involvement of EBNA1-specific T cells in immunopathological processes in MS trough the cross-recognition of myelin antigens .
Although the cross-reactivity hypothesis explains the development of autoreactive cells, it doesn’t seem to be the only cause of the MS onset, as the development of autoreactive cells and antibodies still requires the blood-brain disruption and some inflammation or targeting processes at site of damage ; therefore it seems like some initial local inflammatory damage in the CNS might trigger the activation of cross-reactive and autoreactive myelin-specific T cells. However, the question that that requires further research is what type of CD8+ T cells enters the CNS and causes inflammation and demyelination during MS attacks – EBV-reactive, myelin-specific CD8+ T cells cross-reacting with EBV, or both types of cells.
3.2.4. EBV-infected autoreactive B cells hypothesis
As EBV immortalizes B cells and facilitates their differentiation into long-lived memory B cells, these mechanisms might underlie the survival of autoreactive B cells, which in certain context may also act as an antigen-presenting cells contributing to the development of autoimmune process. M. Pender has proposed the EBV-infected autoreactive B-cell hypothesis of MS, according to the later version of which the following scenario is suggested . CD8+ T cells deficiency and impared control over EBV by CD8+ T cells in patients with MS result in a high frequency of EBV-infected memory B cells, including autoreactive B cells, which infiltrate the CNS and produce oligoclonal IgG in the cerebrospinal fluid. Systemic viral infections, especially in individuals carrying MS-associated polymorphic variants of class II HLA, can lead to the activation of CD4+ T cells that are able to cross-react with CNS antigens. These CNS-reactive CD4+ T cells migrate into the CNS where they are reactivated by EBV-infected B cells presenting CNS antigens. These EBV-infected B cells provide costimulatory signals that promote T cell survival, thus inhibiting activation-induced apoptosis of T cells, which normally occurs when autoreactive T cells infiltrate the CNS. The autoreactive T cells activated in this way organize an immune attack on the CNS by recruiting monocytes/macrophages and B cells. An increasing number of CNS antigens releasing as a result of this attack leads to the spread of an immune response to other CNS antigens. Repeated attacks of T cells on the CNS, supported by local EBV-infected B cells, facilitate the development of meningeal B-cell follicles with germinal centers that generate CNS-reactive B cells producing autoantibodies that cause demyelination and neuronal damage in the cerebral cortex and the cerebellum, leading to a progressive phase of MS. CD4+ T cells activated in the CNS, in turn, can complement the BCR and CD40 receptor signaling on the surface of the EBV-infected B cells, which is already provided by LMP2A and LMP1, respectively, that is, provide a "double signaling". This can lead to a vicious circle in which activated EBV-infected autoreactive B cells promote autoimmunity, which in turn promotes EBV infection of the CNS . Although this scheme complements the previous hypotheses and may explain the rare occurence of MS in general population infected with EBV as well as the contribution of genetical context to the autoimmune process, the question whether B cells in MS brains are autoreactive still needs to be elucidated.
Taking into account the studies of EBV in MS CNS mentioned above, it might be thought that in MS EBV-directed CD8+ T cell cytotoxicity leading to the bystander CNS damage with release of myelin antigens might precede the migration of immune cells including autoreactive CD4+ and CD8+ T cells into the CNS. However, this suggestion has to be verified by additional studies.
3.2.5. Emerging mechanisms: EBV empowers human B cells for autoimmunity
Another recently proposed hypothetical mechanism, potentially involved in the pathogenesis of MS, is related to the presentation of myelin antigens to CD8+ T cells by EBV-infected B cells.
Recent data obtained by Morandi E. et al. have shown that infection of human naïve B cells with EBV upregulated the markers associated with the antigen presentation and switched the processing of human MOG protein in B cells in a way that facilitated cross-presentation of disease-relevant MOG epitopes to CD8+ T cells . Authors suppose that the difference between the prevalence of MS and of EBV infection might depend on the fraction of myelin-specific B cell clones that are infected with EBV . This mechanism seems to complement the previous hypothetical mechanisms of EBV involvement in MS, and it would be very interesting to clear out whether this EBV-induced abnormal switching in myelin processing is associated with certain genetical strains of EBV or represent more widely spread EBV-associated mechanism which becomes detrimental in certain immunological context predisposing to MS.
3.2.6. EBV activates HERV-W/MSRV
Recent studies give evidence for that EBV may also activate human endogenous retroviruses (HERV) [153,154], which have been found to be directly involved in the inflammatory and neuropathological processes in CNS observed in multiple sclerosis. EBV-infected B cells in the CNS also seem to contribute to proinflammatory milieu, as EBV infection activates expression of HERV in B cells [153,154]. This hypothesis is supported by a number of studies and be will be discussed in detail in the section on endogenous retroviruses.
Involvement of other viruses in the pathogenesis of MS
Based on the understanding of the pathogenetic role of EBV in the development of MS, theoretically, the delay between seroconversion in the EBV-positive status and the onset of MS in young people would be short enough because the EBV load is 10,000 times higher during primary infection compared to established EBV infections, in which a transition from a high viral load to a low occurs over approximately one year [71,151]. However, in fact, this delay lasts for several years, which, together with the profound heterogeneity of the disease, reflects the existence of additional factors in virus-mediated pathogenetic mechanisms of MS. In addition to EBV, several viruses have been supposed as cofactors for pathogenesis of MS, in particular HHV-6 and human endogenous retroviruses (HERV).
3.3.Varicella-zoster virus (VZV)
Varicella zoster virus (VZV) is the herpesvirus that causes varicella (chickenpox) as a primary infection during childhood, and late reactivations as zoster in older individuals. By the age of 15 years, 95% of individuals in developed countries have acquired the infection [155,156].Varicella-zoster is neurotropic but for the most part remains dormant in the sensory ganglia. However, immunosuppressive events occurring later in life, such as stress, can result in its reactivation, evidenced by zoster /shingles and, in severe cases, encephalitis. Both the neurotropic potential and the relapsing-remitting nature of infection make this virus an attractive culprit for influencing the natural history of MS . The results of many epidemiological or serological studies failed to confirm the link between VZV and MS; a report evaluating 40 studies in the period 1965–1999 indicated that there is insufficient evidence to support the aetiological role of VZV infection in the development of MS .On the other hand, studies of Sotelo et al. demonstrated an increase in VZV reactivation measured by PCR in PBMCs of 82% patients during clinical relapse, an effect that was not observed in MS patients during remission . These results were confirmed by the study on a larger cohort of patients, in which VZV-specific DNA was found in PBMC in 95% of patients during relapse compared with 17% of MS patients on remission. Flow cytometry analysis carried out in this study also showed that a relapse was associated with a significant percentage of lymphocytes positive for VZV antigens when compared to MS patients during remission or with controls . It was also observed by real-time PCR that the amount of VZV found in PBMC from MS patients during relapse was high during the first week of the acute relapse but became gradually minor on clinical remission, until its disappearance in samples taken 2 months later .
Furthermore, the amount of viral DNA measured in MS patients during relapse was hundreds of times higher in CSF than that in PBMC; in both locations the VZV DNA loads also decreased after the acute relapse . Electron microscopy used in this study showed the presence of VZV viral particles in the CSF from all 15 MS cases studied in relapse . In contrast, another study failed to show the presence of VZV DNA in the CSF or in the acute plaques of MS patients ; furthermore, recombinant antibodies prepared from clonally expanded plasma cells in MS CSF, which are thought to represent the intrathecally synthesized oligoclonal IgG, did not bind to VZV-infected cells .
Taken together, several studies suggest the link between VZV reactivation and the occurrence of MS relapse, but the role of VZV in MS remains controversial, and additional studies required to elucidate whether VZV is an essential environmental trigger of MS and the contribution of this virus to the pathogenesis of MS.
3.4.Human herpesvirus-6 (HHV-6)
Like EBV, HHV-6 infection usually occurs during childhood and persists for the duration of life. However, results of the studies on HHV-6 involvement in MS has been contradictory.
Early studies reporting the presence of HHV-6 virus DNA in the brain and in the CSF of patients with MS and of control group found that HHV-6 has a strong neurotropism to the CNS compartment. This was confirmed by several subsequent studies reporting higher levels of HHV-6 expression in brain samples of MS patients compared to control, and higher levels of viral DNA and mRNA, especially in demyelinated plaques [reviewed in 161]. In contrast, the study of Mameli et al. reported about lack of expression of HHV-6-specific RNAs in analyzed brain samples of MS patients .
In one study the evidence was showed for the cross-reactivity between myelin basic protein (MBP) and HHV-6 in multiple sclerosis , but in another study no significant differences were found between MS patients and the control group regarding the ability of HHV-6 to activate MBP-reactive T cells .
The important fact is that all evidence relating MS concerns HHV-6A, but not HHV-6B, two quite different viruses . HHV-6A is a neurotropic virus that infects astrocytes of MS patients . First suggestions about the link between HHV-6A and MS arose from the immunohistochemical demonstration of viral antigen in oligodendrocytes of MS white matter lesions but not in control brains. Two later studies found HHV-6 mRNA and protein expression specifically in oligodendrocytes . Some studies have also found the higher frequency of HHV-6-specific DNA in MS lesions compared to normal appearing white matter [167,168].
The meta-analysis of Tao C. et al. also provides some evidence for the relationship between HHV-6 and MS, but no studies have shown a causal relationship, and there are a number of discrepancies in the results of the studies. For example, some studies showed a higher incidence of seropositivity and higher IgG titres to HHV-6 in the serum of MS patients compared to control. But in other studies, the similar levels of serum antibodies to HHV-6 in MS patients and in the control group were obtained, which made it difficult to determine the role of HHV-6 at the onset of MS. In addition, no studies have demonstrated an increased immune response to HHV-6 before the onset of MS, unlike EBV . HHV-6 reactivation determined in some MS patients by measuring both viremia and anti-HHV-6 specific antibody levels allowed some authors to suggest its strong correlation with increased disease activity. However, results obtained from others indicate a weak association of HHV-6 reactivation with MS exacerbation, as measured by virus-specific cellular immune responses in the blood and MRI activity .
In the study , a subset of oligoclonal immunoglobulins exhibiting specificity for HHV-6 was detected in only 20% of patients with MS. The results prove that in most MS patients without intrathecal Ig-response to HHV-6 (about 80% in this study) this virus does not participate in pathogenesis of the disease . Pietilainen-Nicklen J. et al. investigated the presence of HHV-6- reactive oligoclonal bands (OCB) in cerebrospinal fluid of patients with demyelinating diseases (mainly MS), and the authors have found that patients with OCB reacting to HHV-6 can be isolated in a separate group that was significantly younger, with significantly higher levels of IgG OCB compared to a group of patients without OCB reactive to HHV-6 . The study of Knox K. et al. has also found that patients with active HHV-6 viremia were significantly younger than did HHV-6 viremia-negative patients . Thus, it can be assumed that in a certain number of patients, HHV-6 is involved in the pathogenesis of MS as a cofactor adding specific features into the complex immunophenotype of the disease.
It is known that progression of demyelinating diseases is caused by an imbalance of two opposing processes: persistent destruction of myelin and myelin repair by differentiating oligodendrocyte progenitor cells (OPCs). If myelin reparation process is impaired and cannot compensate the destructive process, the progressive loss of myelin takes place . Recent study has found that infection of human OPCs with HHV-6A with subsequent expression of the latently-associaed U94 viral protein was sufficient to decrease the migration of human OPCs both in vitro and in vivo. These data raise the possibility that HHV-6A latency in the CNS is not a benign state, but can result in defective myelin repair through impaired recruitment of myelinating OPCs after injury, therefore contributing to the progression of chronic demyelinating diseases. This hypothesis is consistent with both evidence of HHV-6 viral infection but an absence of infectious virions in demyelinated lesions and a failure of OPCs to populate these lesion sites .
In addition, it has been shown that HHV-6A is able to activate latent EBV in B cells; it activates expression not only of EBV-encoded lytic cycle proteins but also proteins associated with the “growth” and transformation program, namely, LMP1 and EBNA-2 . A further link between HHV-6A and EBV involves their induction of expression of the human endogenous retrovirus HERV-K18-encoded superantigen. Such virally induced T-cell responses might secondarily also lead to local autoimmune phenomena..
Therefore, current data do not directly confirm the link between HHV-6 and MS. Additional longitudinal cohort studies measuring both serological and viral parameters both in CSF and peripheral blood are required to verify the association between MS and HHV-6. The role of abnormal immune response to HHV-6 in MS activity has been supported by an increasing number of cohort and laboratory studies . Interestingly, the roles of EBV and HHV-6 are somewhat opposed to each other: EBV is strongly associated with the onset of MS while there is more evidence that HHV-6 is associated with disease activity . Current data allow to suggest that if HHV-6 is involved in MS pathogenesis, 1) it probably plays a side role in viral interactions in complex immunological context that may lead an autoimmune reaction or enhance it, in particular, via activation of latent EBV-infected B cells and promotion of expression of HERV-K18-encoded superantigen; 2) the more important role of HHV-6 in MS seems to be associated with its ability to reduce the migration of OPCs infected with HHV-6 to demyelinated lesions, thus disrupting the remyelination process and aggravating the demyelination in MS.
3.5. Human endogenous retroviruses(HERV)
In addition to EBV, the most consistent and independently confirmed data about involvement in the pathogenesis of MS were obtained for human endogenous retroviruses (HERV), especially members of the W family (HERV-W) [173,174]. HERVs may be considered as an intermediate link between exogenous viruses and genes, as they are the remnants of ancient retroviral infections endogenously transmitted by a multitude of generations over tens of millions of years. Now they represent up to 8% of the human genome. HERVs have the same genetic structure as exogenous retrovituses: two long terminal repeat regions (LTRs) encompassing the coding sequence of the four basic retroviral genes: gag, the matrix and retroviral core; pol, the reverse transcriptase; pro, the integrase; and env, the envelope. To date no HERVs has been shown to be fully able to replicate and produce infectious virions . Under physiological conditions, the majority of these elements are inactive or non-functional due to epigenetic control and deactivating mutations ; despite this, transcription of HERV RNA occurs in many normal human tissues. The levels and pattern of HERV expression may be modulated under pathological conditions, especially in cancer and inflammatory diseases, including inflammatory neurological diseases [176,177].
Three HERV families have been associated with MS: HERV-H, HERV-K, and HERV-W. The strongest association with MS was established for two members of the HERV-W family: MS-associated retroviruses (MSRV), a complete virus with extracellular virions, and ERVWE-1, an element expressing only envelope (env) protein called Syncytin-1. Both env proteins, MSRV env and Syncytin-1, have proinflammatory and superantigenic properties and have been shown to cause neurotoxic effects such as neuroinflammation and neurodegeneration, as well as alterations of the immune system; both have been suggested as co-factors triggering the immunopathogenesis of MS , which will be considered further.
Inflammatory stimuli and viral factors may trigger the activation of HERV in human cells. In vitro, the expression of HERV-W/MSRV and syncytin-1 in blood cells and in astrocytes is upregulated by proinflammatory cytokines and inhibited by IFN-α and IFN-β . Viral infections can also activate HERV expression or de-regulate the mechanisms that normally keep HERV in a “silent” state . In culture, expression of HERV-W/ MSRV genes/proteins was activated by some viruses, such as EBV, herpes simplex virus type 1, or by influenza virus [179; 180].
Independent research groups reported that HERV-W env and pol expression could be detected in serum, peripheral blood mononuclear cells (PBMC) and CSF of MS patients. In 15 studies MSRV/HERV-W env or MSRV/HERV-W pol RNA or protein were found to be increased in serum/plasma or PBMC of MS patients compared to controls .
Activation of HERV-W / MSRV has been established in inflammatory and neuropathogenic processes in MS. In cultured PBMC of MSRV-positive patients, expression of MSRV was upregulated by the action of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-6 (while IFN-β significantly decreased MSRV release) . These pro-inflammatory cytokines, in turn, were overproduced in response to MSRV env by PBMC from MS patients, and were found to correlate with MS severity (EDSS) , thus providing pathogenic feedback loop associated with MSRV.
MSRV env was found to specifically activate monocytes through Toll-like receptor 4 (TLR4) and CD14 and stimulate the production of IL-1β, IL-6 and TNF-α . Moreover, MSRV env can also trigger phenotypic and functional maturation of dendritic cells and enables them with the capacity to support a Th1-like type of differentiation [184,185].
At the brain level, HERV-W env activates Toll-like receptors (TLR4) of oligodendroglial precursor cells, which results in the production of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS), and a subsequent decrease in myelin protein expression. Within chronic brain lesions in MS, HERV-W env was detected in microglia/macrophages near TLR4-expressing oligodendroglial precursor cells . Immunohistochemical detection of HERV-W env protein in postmortem brain samples of MS patients showed its elevated levels only in active lesions in astrocytes and microglia, type of cells which are important modulators of neuroinflammation, and the intensity of staining correlated with the degree of active demyelination and inflammation . These results are consistent with other studies, showing relative accumulation of HERV-W env RNA and protein in brain samples of MS patients, with the most pronounced HERV-W env immunoreactivity in areas of active demyelination [187-189].
ERVW-1-encoded glycoprotein, syncytin-1, appered to be distinct from MSRV env (HERV-W env), but with sequence similarities. A major difference between syncytin-1 and MSRV env is the localization of the protein. While MSRV has been reported to be found as an extracellular virus, syncytin-1 is found intracellularly and on the plasma membrane. Syncytin-1 is overexpressed in brains of individuals with MS, and examination of its copy numbers by PCR techniques revealed a significant increase in DNA and RNA copy numbers in the brain tissue but not in PBMCs, CSF or plasma levels compared to controls [reviewed in 190]. By discriminatory PCR assays, that amplify selectively either MSRV env or syncytin-1, the DNA of MS patients was found to have on average 6-fold more MSRV env copies than controls, while the syncytin-1 copies were almost identical for both . Thus, syncytin-1 overexpression seems to be restricted to the brain parenchyma in MS patients, which excludes its usefulness as a clinical biomarker .
It has been shown that several viruses such as herpes simplex virus, influenza virus, and cytokines such as TNF-α are capable of inducing syncytin-1 expression .
As previously shown for MSRV, MS-detrimental cytokines (TNF-α, IFN-γ, IL-1, IL-6) activate syncytin-1 promoter, whereas the MS-protective IFN-β is inhibitory . Like in case of HERV-W env, immunoreactivity to syncytin-1 was obsreved in activated microglia and astrocytes (but not in neurons and oligodendrocytes) in brain samples of MS patients. Expression of syncytin-1 was found to induce the release of iNOS, which suggested ongoing inflammation. The myelin producing cells of the CNS, oligodendrocytes, are particularly vulnerable to free radicals, and syncytin-1-associated secretion of iNOS was shown to cause oligodendrocytes damage and death .
3.5.3. EBV and HERV in MS: a missing link to the pathogenesis of MS?
It has been shown that in vitro binding of the EBV gp350 protein caused activation of MSRV env and syncytin-1 in B-cells, monocytes, macrophages and in astrocytes, cells that may be involved in MS pathogenesis . Monocytes, particularly after their differentiation to macrophages, appeared to be the most responsive to EBVgp350, expressing even higher levels of HERV-W env than B cells . This finding is concordant with another one, which demonstrated that during infectious mononucleosis EBV promoted the strongest activation of HERV-W/MSRV expression in monocytes compared to other blood cell types . These findings are of particular interest for possible involvement of this effect in MS, as monocytes take part in pathogenesis of MS on different levels. Mononuclear phagocytes, such as activated CNS-resident microglia and macrophages that have infiltrated from the periphery, are predominant type of cells in MS lesions in both relapsing-remitting and progressing type of the disease. The functions of activated macrophages and microglia in neuroinflammation are complex, as they are characterized by functional heterogeneity and involved in wide range of processes, including 1) release of proinflammatory factors causing demyelination, 2) presentation of myelin antigens to the T cells, on the one hand, and 3) myelin debris clearance and remyelination in MS lesions, on the other [192-194]. Increased blood-brain barrier permeability is a characteristic hallmark of the CNS alterations leading to MS, suggesting a causal relationship between inflammatory cell recruitment into the CNS and the blood-brain barrier dysfunction. Activated monocytes easily pass across the endothelia, and could carry the HERV-W across the blood-brain barrier, entering the brain, where the HERV-W env protein could facilitate the pathological processes, inducing inflammation, demyelination and axonal damage. EBV-infected B cells in the CNS also seem to contribute to proinflammatory milieu, as EBV infection activates expression of MSRV env and syncytin-1 in B cells [153,154].
Of particular importance is also the study of Mameli et al., who demonstrated that in peripheral blood mononuclear cells (PBMCs) of patients with infectious mononucleosis an increased expression of MSRV / HERV-W was observed, and a direct correlation was found between anti-EBNA-1 IgG titers and the levels of MSRV mRNA expression. Thus, the data indicate that the two main links between EBV and MS – infectious mononucleosis and high anti-EBNA1-IgG titers – are paralleled by activation of potentially neuropathogenic HERV-W/MSRV .
Since HERV-W is expressed by a high proportion of B cells, it is likely that reciprocal interactions occur within these cells during latency. According to the mechanism proposed by Meier et al., latent EBV infection could contribute to (neuro)inflammation, through the expression of small non-coding RNAs (EBER) that bind to Toll-like receptor 3 and potentially other intracellular receptors .
At the same time, EBV binding to human CD21 on resting B cells triggers the expression of the normally inactive HERV-K18 env protein, which has the properties of the superantigen . Recent findings identified that both EBV and HHV-6 induce expression of the HERV-K18-encoded superantigen and therefore may promote polyspecific T cell activation on periphery which may contribute to the pathological process in MS [107,195,196].
A recent immunohistochemical study has shown that not only B cells, but also astrocytes and microglia in brain sections of patients with MS are infected with EBV [ Hassani A. et al., 2018 ]. How EBV enters astrocytes and microglial cells, at the moment remains unknown, but these findings require further studies for elucidating the contribution of EBV infection of brain astrocytes and microglia to HERV-W-associated neuropathological processes in MS.
Thus, current data allow to suggest that EBV may be the initial viral trigger in pathogenesis of MS, contributing to later activation of HERV, which, on the one hand, may cause proinflammatory context and direct neuropathological effects in the CNS, and on the other hand, promote proinflammatory context on the periphery, with possible polyspecific T cell activation contributing to the immunopathological processes in MS.
3.5.4.HERVs association with MS disease activity, progression and treatment response
MSRV : correlation with disease activity and progression. Studies of a cohort of MS patients from Sardinia, which is known for its relatively high incidence of MS, found a direct parallelism between MSRV positivity / load and MS clinical stages and phases of disease activity. Extracellular HERV-W / MSRV and MSRV-specific mRNA sequences were repeatedly detected in blood, cerebrospinal fluid and brain samples of MS patients, and MRSV presence/load was found to strikingly correlate with the stages and active/remission phases of MS . Moreover, MSRV positivity was found to increase with MS duration and progression, which is concordant with other studies [197,198]. The study of Perron et al. has found that HERV-W env DNA copy numbers in PBMC were significantly increased in chronic progressive MS (SPMS and PPMS) compared to RRMS . Elevated number of copies of MSRV / HERV-Wenv DNA in MS patients clearly correlated with higher EDSS score .
A multicentric study of MS patients and controls from different European areas has shown that in all ethnic groups the presence of MSRV particles in blood and CSF was significantly associated with MS . 10-year blind observational study confirmed that the presence of MSRV in the CSF of MS patients on early stages of disease was associated with a significantly greater rates of disability assessed by Expanded Disability Status Scale (EDSS) and with secondary progression of the disease . Of note, in this study the disease progression to a secondary-progressive type occurred only among the patients who at start had MSRV-positive CSF, which allowed authors to propose the MSRV evaluation as a prognostic biomarker to monitor disease progression in individual patients. The fact that the analysis for MSRV-specific pol sequences in the CSF was positive in 50% patients at onset, in 90% of patients with RRMS and in 100% of SPMS patients  indicate that MSRV seems to be not a causative agent, but a factor inequivocally involved in pathogenesis of MS after initial deregulating immune mechanisms leading to proinflammatory context in the CNS.
HERV and treatment response. In a long-term study of effects of interferon-β therapy on MS patients, it was found that in cases of efficient therapy MSRV load rapidly decreased to levels below detection limits (the earliest effect was detected 48 hours after the first administration of the drug), while strong disease progression with therapy failure was accompanied by MSRV rescue . In interferon-β therapy, accompanied by lowering in disease activity, there is also a significant decrease in the reactivity of antibodies to HERV-W env and HERV-H env . Taken together with the abovementioned studies, these findings speak well for the potential use of blood MSRV as the prognostic marker for the individual patient, to monitor disease progression and therapy outcome.
3.5.5.HERV-Wenv: potential therapeutical implications.
Several experimental studies showed that imunopathogenic and gliotoxic effects promoted by HERV-W env were efficiently neutralized or reversed by a specific antibody to MSRV e nv, which confirms the specificity of MSRV e nv in the observed pathogenicity . The association of HERV-W env with demyelinating cells in MS lesions can support the rationale of a therapeutic strategy using a neutralizing antibody against this protein .
In view of the above findings, attempts to target the neuropathogenic HERV-W env with the use of humanized neutralizing antibody GNbAC1 were undertaken. Phase IIa clinical trials in RRMS patients, a one-year study with monthly GNbAC1 infusions, showed safety of the treatment . After 48 weeks of clinical trials, patients who had been treated continuously with GNbAC1 had brain imaging evidence for benefit on key markers of neurodegeneration, linked to disease progression; safety and tolerability of GNbAC1 remained good. However, the benefit of GNbAC1 for patients with MS seemed to be mainly in slowing of neurodegeneration and possible neuroprotection/remyelination, rather than a reduction of neuroinflammation. Perhaps higher doses of GNbAC1 should be tested in people with MS to see if a more robust effect can be seen . One of the possible explanations of moderate positive effect of this treatment may be the involvement of other viruses, like EBV (and HHV-6 in some patients), in the neuroinflammation in MS, which is worthy of further investigation and development of new therapeutical strategies.
Thus, despite that our current understanding of the context triggering the initial pathological response leading to MS is incomplete, most likely the combination of factors contributes. M. Sospedra suggestsed that these probably include the susceptibility-conferring genetical background as well as an infectious context and strong activation of both innate and adaptive immune systems. This activated state leads to a shift of the immune response towards a proinflammatory Th1 phenotype and may be induced by such conditions as high antigen dose and the presence of proinflammatory cytokines; viral tropism to the target tissue; local tissue destruction and/or immune activation in the target tissue .
4. Conclusions and future perspectives
The involvement of viruses in pathogenesis of autoimmune diseases is complex, may occur through different mechanisms which are likely context-dependent, may occur simultaneously and are not mutually exclusive . Multiple evidence supports the strong association between EBV and MS, including the 4-fold increase in risk of MS in individuals with the history of infectious mononucleosis compared with subjects with asymptomatic primary EBV infection ; a significant increase in IgG titres to EBV antigens, mainly to EBNA-1, in serum of patients a few years before the onset of clinical manifestations of the disease [90-92]. Independent studies consistently reported that nearly 100% patients have been shown to be infected with EBV, with one study indicating 100% EBV-seroconversion prior to MS onset, which allows to suggest that EBV infection as a pre-requisite to MS . Over the past decade convincing data have also been obtained on the likely relationship between acute inflammation, EBV reactivation and cytotoxicity directed toward EBV-infected B cells in the CNS of MS patients. Different studies showed the EBV-directed reactivity of CD8+ T cells in MS brain and CSF samples [84,121,124-130]. Furthermore, some studies have shown the deficiency and exhaustion of EBV-specific CD8+ T cells in MS patients [124,141,142,144,149], and it has been hypothesized that impaired control over EBV by virus-specific CD8+ T cells might lead to uncontrolled increase in number of EBV-infected B cells and allow them to accumulate in the CNS . The promising results obtained in recent phase I clinical trial of in vitro -expanded autologous EBV-specific CD8+ T cells in patients with progressive forms of MS  add to the mounting evidence for a pathogenic role of EBV infection in MS and speak in favor of the necessity of further testing of safety and efficacy of EBV-specific adoptive cell therapy in patients with progressive forms of MS.
The aggravation of EBV-specific CD8+ T cell deficicency leading to accumulation of EBV-infected memory B cells in the CNS may partly explain the delay in several years between the primary EBV infection and MS onset. However, the question that needs to be addressed is why despite that most of the world’s population is infected with EBV, only small percent of individuals develop MS (∼0.1% of the population). The autoreactive B-cells hypothesis proposed by M. Pender [57,72] which could potentially explain such a rare MS occurence requires clarifying whether EBV-infected B cells in MS brains are autoreactive. On a level of EBV genetic variability, a recent study has found that the association between EBV and MS is dependent on genetic variants of EBV, involving especially EBNA2 gene, the most polymorphic region of the EBV genome . Another possibility, which may be associated with a rare occurrence of MS, may be the phenomenon of a random EBV integration into a host cell, which can cause methylation and gene expression changes in B cells and thus may potentially contribute to the pathogenesis of MS . On epidemiological level, the fact that occurence of symptomatic primary EBV infection in Western countries by far exceeds the prevalence of MS (more than 50-fold) suggests that additional genetic and/or environmental factors determine whether symptomatic EBV infection indeed predisposes to MS . Even if EBV is truly the main contributor to MS pathogenesis, the complexity and heterogeneity of MS can not be explained by a single etiological agent and indicate the involvement of additional factors.
According to a significant number of independent studies, among additional viral factors contributing to the pathogenesis of MS, members of the HERV-W family, MSRV env and Syncytin-1, seem to be the most likely candidates due to their proinflammatory and neuropathogenic properties, their presence in MS brain lesions and striking correlation of HERV-W env /MSRV expression with MS activity, progression and treatment response [173,178,181]. It is known that proinflammatory context and viral infections can activate HERV expression or de-regulate the mechanisms that normally keep HERV in a “silent” state . Mameli et al. demonstrated that binding of EBV induce the activation of MSRV / HERV-W in peripheral blood mononuclear cells and astrocytes . In infectious mononucleosis, an increased expression of MSRV / HERV-W by PBMC was observed, and a direct correlation was found between anti-EBNA-1 IgG titers and the levels of MSRV mRNA expression. Thus, the data indicate that the two main links between EBV and MS – infectious mononucleosis and high anti-EBNA1-IgG titers – are paralleled by activation of potentially neuropathogenic HERV-W/MSRV . These data reinforce the hypothesis of research group of Mameli et al. , according to which EBV is the initial trigger of MS, which becomes clinically apparent years later, while MSRV seems to play a direct role of effector of pathogenicity. The mechanisms of activation of HERV-W in latently EBV-infected B cells as well as in EBV-infected astrocytes and microglia  and their contribution to the neuroinflammation observed in MS are worthy of further investigation. Taken together, these findings may open new opportunities for therapeutical interventions against MS.
It is likely that complex interactions between different viruses and host immune system with susceptible genetical backgroung might set the stage for abnormal immunological and neuroinflammatory processes leading to MS. Although there is some evidence for the association between HHV-6 and MS, HHV-6 seems to have a minor role in MS pathogenesis and according to the current data it can be assumed that in a certain number of patients, HHV-6 is involved in the pathogenesis of MS as a cofactor adding specific features into the complex immunophenotype of the disease. HHV-6A was also shown to activate latent EBV in B cells , which may stimulate the proinflammatory context in the CNS, thus contributing to neuropathological process. Some studies on another viral candidate potentially triggering multiple sclerosis, VZV, have shown the presence of this virus in CSF and peripheral blood mononuclear cells of patients with MS in relapse, but further independent studies are required to support the environmental role of VZV in the disease.
Thus, although the most compelling evidence suggests the essential role of EBV as an initial viral trigger of MS and HERV-W – as a direct effector of neupathogenicity, these data don’t exclude the minor contribution of additional viral factors like HHV-6A, VZV or other viruses, which might overlap with individual’s immunological and genetical context, promote proinflammatory state and HERV-W activation, and might explain the profound heterogeneity of disease. Complex interactions between these viruses are likely contribute to the MS pathogenesis and seem to be worthy of further investigation.
Among another questions worthy of further investigation in the framework of the viral hypothesis of MS are: elucidation of immunological and genetic settings predisposing to MS including identification of immunological and genetic factors associated with the defective control of EBV-infected B cells in CIS and MS patients; the pathogenetic context and mechanisms for the migration of EBV-infected B cells to the CNS leading to the development of MS; MSRV / HERV-W - associated pathogenetic mechanisms at different stages of MS development. The latest data on the association of EBV and HERV / MSRV with neuropathogenic processes in the central nervous system are the important basis for the development and improvement of treatment strategies for MS, and one of the perspective directions in this field seems to be the search for therapeutical strategies which would efficiently combine targeting EBV-infected B cells and reducing expression of HERV-W/MSRV.
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1 Mechnikov Institute of Microbiology and Immunology of the National academy of medical sciences of Ukraine, Kharkiv, Ukraine
2 Institute of Dermatology and Venereology of the National academy of medical sciences of Ukraine, Kharkiv, Ukraine