Early disease stages of multiple sclerosis (MS) are primarily characterized by immune cell infiltration of the central nervous system (CNS).
This causes inflammation that damages the myelin sheaths that insulate nerves, which are established by specialized glial cells of the CNS called oligodendrocytes.
These structures protect, nourish and stabilize the axons that transmit electrical signals between neurons.
There is a large therapeutic repertoire of immunomodulatory drugs available that can effectively target the inflammatory aspects of relapsing multiple sclerosis (RMS).
But when MS progresses, damage accumulates, which ultimately results in irreversible deficits and clinical disability.
Unfortunately, despite decades of intense research, disease progression is still untreatable, as there are no therapies available that either prevent damage or repair injured axons.
In a new study published on June 18 in PNAS, a research team led by Professor Dr. Patrick Küry from the Department of Neurology (chaired by Professor Dr. Hans-Peter Hartung) has shed light on a novel axon damage mechanism that could be highly relevant for progressive MS (PMS) patients.
As outlined by first author Dr. David Kremer, the envelope (ENV) protein of the pathogenic human endogenous retrovirus type W (pHERV-W) was found to be a major contributor to nerve damage in MS.
In collaboration with research teams in the U.S. and Canada, the authors demonstrated that the ENV protein drives CNS resident microglial cells to contact and damage myelinated axons.
Alongside the scientific research into the damage mechanism, clinical developments aiming at neutralizing the harmful ENV protein in MS patients have also progressed.
Two clinical studies conducted under the supervision of Professor Hartung have already successfully tested the ENV-neutralizing antibody temelimab.
MRI scans of the participants treated in the study showed reduced damage to the nerve tissue.
The Düsseldorf-based researchers and their colleagues can therefore explain why neurodegeneration is decreased in patients treated with temelimab.
This antibody specifically binds to the ENV protein of the retrovirus and blocks its activity in the CNS. Professor Hartung says future clinical studies in progressive MS patients will now have to demonstrate whether temelimab treatment can also improve clinical symptoms resulting from neurodegeneration.
HERVs, retroviral sequences integrated into the genome during evolution, are now known to represent 8% of the human genome.
These were recently shown to comprise copies that retain potential to express retroviral proteins or particles, and can be abnormally expressed in autoimmune, neurodegenerative, chronic inflammatory diseases, and cancer.
Environmental factors such as specific viral infections were shown to potently activate HERVs under tissue-specific and temporal conditions.
Of several diseases in which abnormal activation and expression of HERV proteins have been reported, studies over recent decades have led to a proof of concept that HERVs play a key role in the pathogenesis of MS and ALS.
HERV-W and HERV-K Env proteins induce pathogenic effects in vitro and in vivo that are relevant to the pathognomonic features of these diseases.
These endogenous retroviruses are potential novel therapeutic targets that are now being addressed with innovative therapeutic strategies in clinical trials.
The causes of multiple sclerosis and amyotrophic lateral sclerosis have long remained elusive. A new category of pathogenic components, normally dormant within human genomes, has been identified: human endogenous retroviruses (HERVs).
These represent ∼8% of the human genome, and environmental factors have reproducibly been shown to trigger their expression.
The resulting production of envelope (Env) proteins from HERV-W and HERV-K appears to engage pathophysiological pathways leading to the pathognomonic features of MS and ALS, respectively.
Pathogenic HERV elements may thus provide a missing link in understanding these complex diseases. Moreover, their neutralization may represent a promising strategy to establish novel and more powerful therapeutic approaches.
Viruses in MS
An infectious origin of MS was suggested for the first time by Pierre Marie in 1884, but was rejected by the contemporary medical community.
The evidence that viruses may contribute to MS comes from the accumulation of immune cells within the brain and CSF, local immune reactivity to specific viruses, and the presence of oligoclonal bands.
MS epidemiology indicates some triggers during adolescence, an unusual geographic distribution (a gradient with latitude, but with exceptions: e.g., contrasting patterns in Sardinia and Japan), and epidemic clusters in previously isolated islands.
Over time, several viruses have been proposed as causative agents of MS. From the 1940s onward they included rabies virus, herpes simplex virus (HSV), scrapie prion, an unidentified MS-associated agent, parainfluenza virus 1, measles virus, simian virus 5, chimpanzee cytomegalovirus, coronavirus, EBV, an unidentified SMON-like virus (subacute myelo-opticoneuropathy virus), tick-borne encephalitis virus, HTLV-1, HSV-1, VZV, and HHV-6.
The proposed mechanisms were direct brain or peripheral infection, activation of autoreactive T cells against nerve myelin, bystander activation, epitope spreading, molecular mimicry, and virus–virus interactions.
However, the link to MS was shown to be weak for the majority of the above viruses.
The most consistent and independently confirmed studies for viral involvement in MS are for EBV.
They appear to be confirmed, but only with indirect links, by history of infectious mononucleosis (IM; primary infection with EBV causes IM), and high anti-EBNA-1 (EBV nuclear antigen 1) IgG titers before MS onset. However, a new concept arose with the discovery that HERVs express pathogenic proteins in disease, and the best evidence of an association and pathogenic involvement is for HERV-W/MSRV (detected in MS blood, spinal fluid, and brain, in parallel with MS stages, active/remission phases, and therapy outcome). EBV is known to activate HERV-W/MSRV in vitro and in vivo (in IM patients and in healthy humans with high anti-EBNA1 IgG titers).
This suggests that EBV could be an initial trigger, and that HERV-W/MSRV is a direct neuropathogenic contributor, before and during MS, in addition to its known contribution to promoting autoreactive T cells, immunoinflammation, and remyelination blockade.
Endogenous Retroviruses Originate from Ancestral Germline Infections by Exogenous Elements
The eukaryotic genome is composed of a large set of DNA sequences, many of which derive from mobile genetic elements [1, 12]. These were estimated to account for about 50% of the human genome [13, 14, 15] (Figure 1A), if not more [16]. Their detection is particularly complex, which may explain why their proportion within the genome has been largely underestimated and why data evolve with technological improvements [12], among which are those allowing the sequencing of constitutive heterochromatin regions that long remained inaccessible [17].
Two types of transposable elements can be distinguished: (i) elements that can be transposed via a DNA intermediate and a cut-and-paste mechanism (transposons) [18], and (ii) those using a RNA and a copy-paste mechanism (retrotransposons) [16]. Retrotransposons comprise endogenous retroviruses (ERVs), for which the current literature presents incomplete data as well as several classifications and nomenclatures, leading to ambiguities [15].
The term ‘HERV’ refers to the sequences of human ERV families.
HERVs retain structural features of retroviral genomes (Figure 1B), and originally entered the genome of species through repeated infections of germ cells over millions of years (Figure 2) [5, 19].
Silent HERVs Can Be Activated by Environmental Triggers
The question of why and how HERV copies that have retained coding potential may become expressed is now addressed. The ability of HERVs to become activated is linked to the chromatin state where a given copy is located [20].
DNA methylation and histone modifications are essential to the epigenetic control of human genes, including HERV elements.
The prerequisite for functional transcription is that HERV sequences must retain functional long terminal repeats (LTRs), or become controlled by another promoter, without deletions or nucleotide substitutions that disrupt their open reading frames (ORFs).
Because the nuclear microenvironment including the chromatin accessibility of coding regions differs between tissues, the baseline predisposition of a HERV copy to be activated can be tissue-, cell-, or maturation stage-specific.Inflammatory stimuli may activate HERVs via epigenetic dysregulation.
For instance, transcription of HERV-W sequences has been reported to be upregulated by proinflammatory cytokines in cultured cells from MS patients [21], and this was shown to correlate with increased differentiation of peripheral blood mononuclear cells from MS patients [22].
By contrast, reductionof anti-Env antibody reactivity for HERV-H and HERV-W [23] and MS-associated retrovirus (MSRV) load [24] in the blood have been in seen in MS patients treated with IFN-β, suggesting efficacy of the therapy or low disease activity.
After an initial study, which first presented evidence that herpesviruses might trigger HERVs in patients with MS as part of the retrovirus hypothesis for MS etiology [25], several teams showed that transcription of HERV genes and/or reverse transcriptase (RT) activity is increased in various human cells in vitro by Herpesviridae with tropism for nervous cell.
This includes herpes simplex virus type 1 (HSV-1) in lymphocytes from MS patients [26] and in neuronal or in brain endothelial cell lines [27]; varicella-zoster virus (VZV) in lymphocytes from MS patients [26]; cytomegalovirus (CMV) in kidney transplant recipients [28]; human herpes virus type 6 (HHV-6) in lymphocytes from MS patients [26] and in T cell leukemic cell lines [29]; and Epstein–Barr virus (EBV) in T cell lines [30] and in peripheral blood mononuclear cells from MS patients as well as in astrocyte cell lines [31].
Many of these viruses have been implicated in MS (reviewed in [32]).
Herpesviridae may invade brain parenchyma and induce local proinflammatory responses, but these pathogens are normally intercepted by perivascular macrophages (Figure 3).
Macrophages are not permissive for their replication but may allow expression of HERV-transactivating Herpesviridae immediate-early genes [33].
The EBV gp350 protein has been shown to activate HERV-W in vitro in B cells and monocytes, but not in T cells, whereas monocyte/macrophage cells appear to be most susceptible [31].
Because EBV has also been shown to potently modify epigenetic traits of host cell DNA (reviewed in [34]), the reported association with infectious mononucleosis, in addition to the elevated anti-EBV nuclear antigen-1 IgG titers in patients with MS [35], might support the hypothesis that EBV acts as a priming trigger. However, this has not been directly demonstrated.
Nevertheless, Herpesviridae (or other environmental activating factors) are now suggested to upregulate HERV-W expression, with its Env protein acting as a pathogenic effector in MS [36].
Other viruses reported to transactivate HERVs are the exogenous retroviruses HTLV-1 and HIV-1.
Specifically, the HTLV-1 Tax transactivator potently increased the transcriptional activity of HERV-W, HERV-H, HERV-K, and HERV-E families in T cells [37].
Moreover, effects of HIV on HERV-K and on HERV-W in astrocyte cell lines and peripheral blood cells in vitrohave been reported to be mediated by the HIV Tat protein, which can indirectly activate HERV-W through Toll-like receptor-4 (TLR4) along with TNF-α and NF-κB.
Thus, by this pathway, HIV Tat could influence non HIV-infected cells [22].
This indirect mechanism suggests that the HIV-driven activation pathway requires persisting Tat stimulation for HERV activation.
This differs from Herpesviridae in that self-sustained HERV expression may be induced following specific triggering events, which could explain the lifelong progression of MS and the multiple triggering events that are required before a pathogenic threshold leading to disease onset can be passed.Nonetheless, if HERV proteins are not expressed, HERV RNA expression (transcription alone) does not seem to have biological effects per se in humans.
Moreover, when produced, HERV proteins are not implicitly pathogenic.
An example is provided by the gag (group-specific antigen)-encoded capsid protein of HERV-W which had no immunopathogenic effect on peripheral blood lymphocyte cultures from healthy donors, whereas the Env protein of HERV-W particles (previously termed MSRV) induced proinflammatory and superantigen (SAg)-like effects [38].
Immune Cells Can Mediate Major Effects of Pathogenic HERV Expression
Abnormal activation of some HERVs is thought to have proinflammatory effects, leading to dysregulation of the immune system, as we will now illustrate with relevant examples.HERV-K and HERV-W families share an interesting common feature in that the protein encoded by their env gene has been shown to trigger responses in T lymphocyte cells expressing a specific variable region of the T cell receptor (TCR) β chain in vitro [30, 38].
Usually, T lymphocytes recognize their target antigen through a combination of variable domains in TCR chains that define the antigen-binding site of specific T cell clones.
The interaction of these HERV Env proteins with another TCR region, which is known to be independent of the antigen-binding site and present on numerous T cells, is known to activate multiple clones irrespective of their antigen specificity.
Molecules inducing such polyclonal activation are SAgs. An inflammatory loop also likely contributes to HERV pathogenicity: following initial priming and induction of specific HERV copies, macrophages and/or B cells produce HERV Env proteins that might fuel local innate inflammation and upregulate major histocompatibility complex (MHC) expression, causing further polyclonal activation of tissue-attracted T cells and therefore of B cells.
This potential inflammatory loop could result in the development of devastating local lesions, while antigen-presenting cells stimulated via TLR and polyclonal activation of lymphocytes might promote breaks in immune tolerance that lead to autoimmunity, as was observed in a mouse model of experimental autoimmune encephalomyelitis (EAE) treated with HERV-W Env protein [39].
HERV-W and MS
MS is an inflammatory disease of the central nervous system (CNS) and a major cause of neurological impairment in young adults.
There is no available cure for MS, and current therapies can only limit the number of relapses and slow disease progression.
The most common symptoms include chronic fatigue, paresthesia with acute and chronic pain, optic neuritis, paresis, gait disturbance, incoordination, sphincter problems, and cognitive impairment, altogether leading to progressive disability.
Histopathologically, MS is characterized by demyelinating lesions that predominantly expand in the white matter, causing destruction of myelin and oligodendrocytes and leading to axonal disruption in the brain and spinal cord.
Lesions spread to cortical regions, affecting grey matter and neurons.
Active lesions also show blood–brain barrier (BBB) breakdown, with infiltration of macrophages and lymphocytes, whereas activated microglia represent the hallmark of regions within active and chronic lesions, where ongoing demyelination and axonal loss are now understood to cause functional impairment (reviewed in [40]).
Immunological dysfunctions in MS are characterized by multifocal CNS hyperinflammatory reactions, and systemic autoimmune reactivity of B and/or T cells towards myelin autoantigens, with evidence of intrathecal chronic IgG production as revealed by the presence of oligoclonal bands in the cerebrospinal fluid (CSF) [41].
Whereas initial disease stages may present reversible phases with remission following relapse, evolution towards progressive forms generally follows [42].
The relapsing forms are dominated by aberrant inflammatory responses, whereas in progressive stages neurodegenerative features take precedence.
This may be paralleled by repeated abnormal lymphocyte stimulation that is known to lead to T cell exhaustion, anergy, or depletion [43], and which implicitly downregulates T cell-driven activation of B cells [44].
The underlying etiology of MS is still not fully understood, but multiple disease-associated loci confer genetic predisposition to develop MS [45], and numerous environmental factors (e.g., infectious mononucleosis and smoking) [46, 47] appear to contribute to disease onset and progression.
This led to the formulation of a pathogenic concept based on gene–environment interactions [48] with a partial, but elusive, role of viral infections [49].
In the search for etiological factors in MS, HERVs have been detected within the human genome, thus opening a new avenue of research because of their potential interactions with environmental factors.
Diverse scientific and technical approaches have indicated that three human endogenous retroviruses, HERV-H, HERV-K, and HERV-W, may be abnormally represented or expressed in MS.A genetic polymorphism in a single copy of HERV-Fc1 (a HERV-H-related element) and its relative distribution in MS patients, with the exception of primary progressive forms (PPMS), was reported [50, 51].
HERV-K mRNA expression was found to be upregulated in postmortem brain tissue from MS patients [52], but no evidence of protein expression was provided.
Retroviral sequences from RT-PCR with selected primers on particles produced by MS derived EBV-B lymphoblastoid cell lines identified sequence variants homologous to HERV-H elements (formerly named RTVL-H or RGH) [53].
Another study showed that B cells and monocytes from patients with active MS showed detectable surface expression of both HERV-H Env and HERV-W Env[54].
Taken together, this evidence suggests that activation of multiple HERV families might be linked to MS [55].
The most compelling evidence for an association between HERV expression and MS is for HERV-W and comes from a recent meta-analysis [56].
This line of research was prompted by the isolation of retroviral particles from MS patients in the early 1990s [57, 58].
Subsequent studies over the following 25 years revealed that these particles originated from HERV elements, first termed MS-associated retrovirus (MSRV), whose sequences were determined from purified retrovirus-like particles isolated from MS cell culture supernatants [59, 60].
The initial sequence identified from several MS isolates was obtained using a PCR protocol designed to detect unknown retroviral sequences flanked by conserved domains in most retroviral pol (polymerase) genes [61].
This prototype MSRV sequence identified a previously unknown HERV family, now named HERV-W because it uses a tryptophan (W) tRNA as a primer for reverse transcription, and that comprises multiple copies homologous to MSRV prototypic sequences [59, 62].
Independent groups subsequently reported that HERV-W env and pol expression could be detected in serum, peripheral blood mononuclear cells (PBMCs), and CSF from MS patients but not from healthy controls [60, 63, 64, 65, 66].
HERV-W association with MS was further evidenced by studies showing that expression levels not only correlated with MS but also increased with disease activity and progression [24, 64, 65, 67].
Further investigations indicated that HERV protein expression is mainly restricted to macrophages and microglia, a minor proportion of lymphocytes, and to a few endothelial and astrocyte cells in active lesion areas [31, 63, 68, 69].
HERV-W Env protein was similarly detected in MS lesions by using different monoclonal antibodies (mAbs) directed against different epitopes [64].
Its association with areas of active demyelination from active to chronic brain lesions, with fairly intense expression until the death of the patient, as seen post-mortem, suggests an involvement in the long-term pathogenic progression of the disease [69].
HERV-W was further revealed to play functional roles in inflammatory processes. Proinflammatory cytokine expression was shown to be induced in both human and murine monocytes upon in vitro stimulation with HERV-W recombinant Env protein, a process that required TLR4 receptor activation [39, 70, 71], while MSRV Env-treated human dendritic cells were elicited to promote type 1 T helper cell (Th1)-like lymphocyte differentiation [71].
MSRV particles (HERV-W) were previously shown to induce superantigen-like T cell responses that were reproduced by the Env protein but not by the gag-encoded capsid protein [38].
A hypothetical scenario illustrating all these effects is presented in Figure 3.
HERV-W envelope also promoted EAE in mouse and induced elevated autoimmune T cell reactivity [39].
In addition to pathogenic effects targeting immune functions, HERV-W Env protein was also found to mediate TLR4-dependent effects on non-immune cells.
Treatment of cultured primary oligodendroglial precursor cells (OPCs) with HERV-W Env was shown to result in an overall reduction of oligodendroglial differentiation via activation of TLR4 [68].
OPCs contribute to neuroregeneration and myelin repair processes in the adult CNS, and blocking their differentiation by HERV-W Env protein may therefore result in remyelination failure (Figure 4).
The negative impact on myelin synthesis in primary oligodendroglial cells could be rescued using a specific Env-neutralizing humanized immunoglobulin termed GNbAC1 [72].
Different mAb versions of GNbAC1 have been assessed on a small scale in MSRV Env-induced experimental allergic encephalitis (EAE), an animal model of MS, and these appeared to inhibit and somehow reverse EAE clinical evolution [73].
In addition, exposure of the HCMEC/D3 brain endothelial cell line to HERV-W Env was shown to impair endothelial cell physiology by boosting ICAM-1 expression (allowing T cell homing into tissues) and proinflammatory cytokine release via TLR4 activation, thereby suggesting that Env might affect BBB integrity (Figure 3) [74].
Together, these data argue that pathogenic HERV-W Env protein (formerly MSRV-Env) is a potential therapeutic target in MS, and that the neutralizing humanized antibody GNbAC1 therefore warrants development; indeed, several early-phase trials have now been completed [73, 75, 76, 77].
A Phase IIb multicenter clinical trial including 260 relapsing-remitting MS (RRMS) patients in 12 European countries is ongoing with this humanized IgG4 antibody.
This trial is a 1 year study with a placebo arm and three treated groups receiving intravenous infusions every 4 weeks (6, 12, and 15 mg/kg) (ClinicalTrials.gov identifier: NCT02782858) [78].
Another approach, based on the hypothesis that antiretroviral drugs that are effective against exogenous HIV infections might also block HERV expression in MS [79], has led to a dedicated evaluation (ClinicalTrials.gov identifier: NCT01767701), but gave negative results for its primary endpoint (http://onlinelibrary.ectrims-congress.eu/ectrims/2016/32nd/146288/julian.gold.phase.2.baseline.versus.treatment.clinical.trial.of.the.hiv.drug.html?f=m3).
This trial was a baseline-versus-treatment study with 20 patients with active RRMS defined as gadolinium-enhancing lesions on magnetic resonance imaging (MRI) at baseline.
They were monitored for 3 months with monthly MRI and then treated with the integrase inhibitor raltegravir for 3 months.
This trial did not reach its primary endpoint goal of significantly reducing either lesion count or lesion development during the treatment period versus baseline.
Nonetheless, the tested drug is a known and effective inhibitor of HIV integrase which plays a role in chromosomal retrointegration of newly generated HIV DNA copies.
Because this may not be a major aspect of HERV expression, and is unlikely to be a key issue for HERV Env production, the mode of action of this anti-HIV drug may not have had relevant effects on HERVs, thus explaining the negative results of the study.
Finally, following a study in which HERV-W Env protein was detected in sera and lymphoid cells of MS patients, but not in healthy controls nor in other neurological diseases, except for a few CIDP cases [64], a recent study confirmed upregulated HERV-W expression in blood cells and peripheral nerve lesions of patients suffering from CIDP [80].
This investigation also showed TLR4-mediated effects of HERV-W Env protein on primary human Schwann cells.
Such direct effects on Schwann cells, and the ability of HERV-W Env protein to cause tissue inflammation and systemic autoimmunity, make it an interesting potential new target for the treatment of patients with CIDP and HERV-W upregulation.
This evidence suggests that HERV-W is not specific for MS, but may be involved in different diseases through the pathogenic properties of its Env protein upon activation by variable factors in different conditions, tissues, and organs.
More information: David Kremer et al, pHERV-W envelope protein fuels microglial cell-dependent damage of myelinated axons in multiple sclerosis, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1901283116
Journal information: Proceedings of the National Academy of Sciences
Provided by Heinrich-Heine University Duesseldorf
