What if the missing ‘environmental’ factor in some of our deadliest neurological diseases were really written in our genome?
Writing in Frontiers in Genetics, researchers from the University of Dusseldorf explain how viruses ended up in our DNA – and what puts them in the frame in unsolved diseases like multiple sclerosis.
The enemy within
A whopping 8% of our DNA comes from viruses.
Specifically, ones called retroviruses – not because they’re old, but because they reverse the normal process of reading DNA to write themselves into their host’s genome.
Retroviruses are old though: they began merging with our earliest, primordial ancestors millions of years ago.
Over the millennia, most of their remnants in our DNA – known as human endogenous retroviruses or HERVs – have been silenced by mutations.
Others, which had evolved to fend off rival viruses, formed the prototypical immune system and to this day protect us from infection.
However, HERVs might also be the missing causative link in major ‘unsolved’ neurological diseases.
“HERVs have been implicated in the onset and progression of multiple sclerosis [MS], amyotrophic lateral sclerosis [ALS] and schizophrenia [SCZ],” says senior author Prof. Patrick Kuery.
“Dormant HERVs can be reactivated by environmental factors such as inflammation, mutations, drugs, or infection with other viruses, so could provide a mechanism for their well-established epidemiological link to these disorders.”
Role in MS
So far, the strongest evidence links HERVs to MS.
“MS is caused by direct autoimmune attacks on myelin – the fatty coating of nerve cells – in the brain and spinal cord,” explains Kuery.
“But we don’t yet understand how these attacks are triggered.”
A variety of studies suggest that reactivation of HERV could be just such a trigger.
“Retroviruses were first associated with MS in 1989, but only decades later was it realized that these are in fact HERVs.
“Subsequently, it was shown that levels of HERV RNA and protein – the ‘readouts’ from reactivated HERV DNA – are increased in the brain and spinal cord fluid [CSF] of sufferers, as well as in their brain tissue postmortem.
“Linking this HERV reactivation to autoimmune attacks in MS, it was found that HERV proteins can trigger an immune response against myelin, which triggers MS-like disease in mouse models.”
Mechanistically, HERV proteins could trigger autoimmunity through ‘molecular mimicry’.
“In addition to direct effects of HERV on myelinating cells, several groups report structural similarities between HERV and myelin oligodendrocyte glycoprotein – a molecule displayed on the surface of myelin.
This similarity could fool the immune system into damaging myelin, when it mounts an attack on HERVs.”
Experimental proof in humans
Similar experiments have linked HERVs to the peripheral demyelinating disease CIDP, as well as more distinct disease processes like progressive loss of motor neurons in ALS (Lou Gehrig’s disease).
In schizophrenia, a complex neurodevelopmental disorder, the link to HERVs is more circumstantial.
“HERV proteins have been reported to increase expression of schizophrenia-linked genes in cultured human brain cells,” reports Kuery.
“However, studies on schizophrenia sufferers show inconsistent changes in HERV expression in blood, CSF and postmortem brain tissue compared to healthy controls.”
Whether or not HERVs contribute to these and other unexplained neurological conditions requires further investigation.
An important step will be to test the effects of HERV-neutralizing antibodies in humans.
“Of note, in relapsing MS patients a phase 2b clinical trial using HERV protein-neutralizing antibody Temelimab has been conducted.
We’re now waiting to see if the treatment showed beneficial effects on remyelination or attenuated neurodegeneration,” Kuery concludes.
Human endogenous retroviruses (HERVs) constitute ∼8% of the human genome, distributed in ∼700 000 elements .
They are remnants of retroviral germline infections that resulted in chromosomal integration into all the cells of the progeny, but their viral replication is defective in the present-day human genome .
Most HERVs are highly degenerated through evolutionary pressure due to accumulation of mutations or homologous recombination between proviral long terminal repeats (LTRs) .
The fixation of HERVs in the human genome is not the end of the immunological conflict between the host and retrovirus.
HERVs’ retroviral origin, together with the fact that they constitute a large amount of the human genome, makes them interesting from an immunological perspective .
By mimicking viral infections, HERVs could function as an ‘intrinsic adjuvant’, possibly sensitizing cancer cells for immune recognition .
Tumor-associated reactivation of HERVs has been reported in cancer tissues , and HERVs may play a prominent role in cancer immunogenicity, either by activating the viral defense pathway (viral mimicry) [4, 5] or by forming a novel pool of tumor-associated antigens that can serve as T-cell targets on tumor cells (Table 1).
The interactions between HERVs and the adaptive immune system could be important for exploiting HERVs as targets in anticancer immunotherapy.
However, some HERV elements, the HERV-encoded Envelope (Env) proteins, have immunosuppressive properties that are important in fetomaternal tolerance  and the mechanism could potentially be used to shield tumor cells from the host’s immunological attacks .
In this review, we discuss how HERVs interact with the adaptive immune system with a particular focus on T-cell responses.
In particular, CD8+ T-cell responses are of particular importance in the defense against viral infection, which HERV expression could mimic, and they are key mediators of cancer cell destruction in cancer immunotherapy.
For HERV annotations, the revised nomenclature by Mayer et al.  was used in this review whenever the primary literature provided sufficient information to support the use of this nomenclature.
Cytotoxic T-cell responses against HERV-derived epitopes reported in the literature
|ERV-4||FLHKTSVREV||6q15||A0201||Renal clear cell carcinoma|||
|ATFLGSLTWK||6q15||A11||Renal clear cell carcinoma|||
All the reported cytotoxic T-cell responses are found in malignant tissues.a
Minor response reported.b
HERV-K MEL is an antigenic protein that is encoded by a short open reading frame from a processed ERVK3.
Adaptive immune response to HERVs
The majority of HERVs have degenerated proviral genomes, which prevent transcription of their retroviral genes.
Still, some HERVs have retained their open reading frames (ORFs) within the human genome with the potential to allow transcription and translation, and hence contribute their viral proteins to the human proteome .
While the level of HERV expression in healthy human tissues is limited , it may be induced during malignant transformation or due to epigenetic therapy.
Since these proteins have retroviral origins, they may form antigens that can be recognized by T cells and B cells.
Animal and human studies have demonstrated both humoral and cell-mediated immune responses against ERV antigens [8–10], which could indicate that immunological tolerance to HERVs is incomplete; however, these antigens’ thymic expression is still poorly understood, and consequently the level of tolerance toward such antigens remains to be determined.
Presentation of HERV-derived antigens by the major histocompatibility complex (MHC) class I is of particular interest to determine the potential immune recognition of HERVs. CD8+ T cells specific for HERV-derived epitopes have been isolated and expanded from the blood of patients with regressing renal cell carcinoma (RCC) following nonmyeloablative allogeneic hematopoietic stem cell transplantation.
Such HERV-specific T-cell mediated the killing of patient-derived tumor cells in vitro .
Rolland et al.  demonstrated that the ERVW Env could induce dendritic cell maturation through an innate immune mechanism, which resulted in a TH1-like immune response. TH1 immune responses are important in the defense against viral infection, which is mimicked by HERV expression.
In the literature, several specific CD8+ T-cell responses against HERV-derived antigens in cancer have been reported (Table 1), indicating the potential importance of HERVs as a target for cell-mediated cytotoxicity in cancer immunotherapy.
Intuitively, humoral immune responses would not be of great importance since HERV expression is intracellular; however, the retroviral Env proteins are transported to the cell surface and could serve as direct targets for humoral immune responses .
Antibody responses may also be driven by inflammatory cell death and uptake, and both humoral and cell-mediated immunity against the ERVK Env protein have been detected in breast cancer patients .
Antibody-mediated immune responses were also reported in ovarian cancer patients and reverse transcriptase activity was observed in the plasma of ovarian cancer patients .
To mount an antibody-driven immune response, either direct secretion of HERV antigens from tumor cells or uptake of dying tumor cells with HERV expression should take place .
In many cases, antibody immune responses are accompanied by a CD8+ T-cell response.
The lytic potential of such T cells may explain the potential shedding of HERV antigens from dying tumor cells and the presence of reverse transcriptase activity in plasma, as observed in ovarian cancer patients .
In particular, antibody responses against the retroviral group-specific antigen (Gag) and Env proteins have been reported in the literature, which could indicate that these viral proteins are highly immunogenic.
Immune tolerance to HERVs
Expression of HERVs in the thymus is not well described, though the intrathymic expression of HERVs is believed to be incomplete mainly due to epigenetic silencing .
It has been speculated that HERVs share strong similarities with exogenous retroviruses and therefore incomplete immunological tolerance could be necessary to defend against exogenous retroviral infections ; however, the basal expression of HERVs in healthy tissues [6, 15] could indicate the need for tolerance toward HERVs to avoid autoimmune responses.
Hence, peripheral tolerance and ignorance mechanisms may play prominent roles in the control of HERV-specific T-cell recognition in healthy individuals .
HERV activation of innate receptors, which mimics viral infections, can potentially induce an inflammatory state, leading to the production and secretion of T-cell-activating chemokines and cytokines [4, 5, 16] and thereby bypass the potential immunological ignorant state. CD8+ T-cell responses against HERV-derived antigens have been reported in the literature (Table 1), strongly indicating an incomplete clonal deletion of HERV-antigen-specific T cells in the thymus. Understanding the differences in HERV expression in the thymus and peripheral tissues would be of great importance for the use of HERVs as immunotherapeutic targets.
T-cell responses against HERVs in tumors
Schiavetti et al. was the first to report a CD8+ T-cell response against an HERV-derived epitope in a melanoma patient. They identified in ERVK3 (HERV-K MEL), a translated region encoding the peptide sequence (MLAVISCAV) presented in HLA-A2 and verified CD8+ T-cell recognition of autologous tumor cells targeting this peptide-MHC complex. ERVK3-specific CD8+ T cells were found in two melanoma patients where the autologous tumor cell lines expressed ERVK3, but no ERVK3-specific CD8+ T cells were detected in the blood of three healthy individuals analyzed in parallel .
This study was followed by Rakoff-Nahoum et al. who, in silico, predicted 15 peptides from ERVK Gag for prevalent HLA alleles and divided these peptides into four pools for an initial screening of T-cell responses in patients with seminoma.
T-cell responses to the ERVK peptide pools were found in the peripheral blood mononuclear cells (PBMCs) of seminoma patients at a much higher frequency than in healthy controls .
Some cancer types seem to have high and preferential expression of certain HERVs; in RCC, ERVE-4 has been shown to be prominently and preferentially expressed in comparison to other tumor types and to normal tissues, where no expression is observed [6, 19].
The long-conserved ORFs in ERVE-4 envcould indicate a retained capacity to produce partial Env proteins in RCC tumors .
In several studies, HERV-specific T-cell recognition was detected in RCC.
From the ERVE-4 locus, four epitopes have been identified that give rise to specific cytotoxic T-cell responses [11, 19]. Of these, Cherkasova et al. show that three HLA-A0201 in silico-predicted peptides from the putative ERVE-4 Env protein could expand CD8+ T cells in vitro .
Takahashi et al. reported a regression of metastatic RCC in patients following nonmyeloablative allogeneic hematopoietic stem cell transplantation with the detection of tumor-reactive, donor-derived CD8+ T cells in the blood of the patients. With the use of cDNA cloning, they identified an epitope from ERVE-4 as responsible for the tumor-reactivity of the donor-derived CD8+ T cells .
T-cell recognition of HERVs has likewise been described in a few other cancer types. Mullins and Linnebacher demonstrated the presence of T cells that reacted to the ORF of ERVH-2 in patients with gastrointestinal cancers. ERVH-2 has previously been shown to be strongly expressed in a subset of gastrointestinal cancers while having low expression in other tumor types and healthy tissue.
They further used 10 ERVH-2-derived peptides for the in vitro stimulation of T cells from healthy donors and demonstrated the induction of ERVH-2-specific T cells capable of lysing a HLA-A0201 colorectal cancer cell line expressing ERVH-2 .
Also in breast cancer patients, T-cell responses against ERVK were demonstrated following stimulation of PBMCs with autologous dendritic cells pulsed with ERVK Env antigens. T-cell proliferation was observed together with cytokine secretion, that is IFN-γ, interleukin-2 (IL-2), IL-6, IL-8, and chemokine CXCL10, only in breast cancer patients, indicating the presence of ERVK Env T-cell recognition in response to disease.
Moreover, T cells with lytic capacity toward ERVK Env antigen were found in breast cancer patients but not in healthy females .
Together, these studies summarized in Table 1 suggest that HERVs can be immunogenic and have the ability to activate adaptive T-cell responses capable of tumor cell recognition, particularly the env gene, which has been suggested to be highly immunogenic.
This antigen could be a promising candidate for future immunotherapeutic strategies such as cancer vaccines; however, among the group of HERVs, there may be substantial differences in their biological effects, potential roles in immune sensitization, and ability to form an antigen reservoir.
Early data point toward distinct features for the expression profiles of different HERVs , but these characteristics remain to be fully elucidated.
Reactivation of HERVs in cancer
The elevated expression of distinct HERVs observed in cancer cell lines could indicate a potential oncogenic role of HERVs, but the causative involvement of HERVs in cancer remains controversial (review the potential oncogenic role of HERVs in Kassiotis ).
A study by Rooney et al.  mapped the 66 HERVs from Mayer et al. to RNA-sequencing (RNAseq) data from 18 different tumor types from The Cancer Genome Atlas (TCGA) and corresponding healthy tissue controls from the Genotype-Tissue Expression project and TCGA.
By mapping the RNAseq data to the HERV transcripts, they demonstrated that numerous HERVs showed enhanced transcription in tumor tissues compared with healthy tissues. Out of the 66 HERV loci, they highlighted three HERV loci (ERVH-5, ERVH48-1, and ERVE-4) with minimal to undetectable transcription in normal tissues and highly elevated expression in certain tumor tissues.
Hence, the authors characterized these as tumor-specific endogenous retroviruses . ERVH-5 is especially prominently expressed in bladder, colorectal, head and neck, lung squamous, ovarian, stomach, and uterine cancers and ERVH48-1 is especially prominently expressed in bladder cancer and prostate cancer, whereas ERVE-4 can be found specifically in RCC. Interestingly, cytotoxic T-cell activity against ERVE-4 was reported previously in RCC [11, 19].
From the Rooney et al.  data, it can be deduced that distinct HERV loci exhibited tumor-associated rather than tumor-specific transcription, because basal HERV expression is observed in healthy tissue, depending on the HERV loci and tissue type.
The Rooney et al.’s study is, to date, the most comprehensive analysis of HERV expression in different cancer tissues compared with healthy tissues. ERVE-4 expression in RCC is a well-described example of tumor-associated HERV expression.
Inactivation of the tumor suppressor gene von Hippel-Lindau results in the overexpression of hypoxia-inducible transcription factor-2α (HIF-2α). HIF-2α binds to HIF response elements, which are located in the 5’LTR of ERVE-4. This LTR was found to be hypomethylated in RCC tumors that expressed ERVE-4, compared with other tumors and normal tissues, indicating a tissue-specific epigenetic regulation in RCC [27, 28].
Cancer-specific transcription factors can be important for tumor-associated expression, for example ERVK has been demonstrated to be activated by the melanoma-specific transcription factor (MITF-M) [14, 29].
ERVK LTRs are additionally susceptible to stimulation by hormones, and hence ERVK expression has been suggested to be relevant in cancers of hormone-regulated tissues .
The tumor-associated expression of HERVs observed in cancer tissues has naturally given rise to one question: do HERVs have a causal role in the development of cancer or is their expression a byproduct of malignant cellular transformation, such as global DNA hypomethylation?
Global DNA hypomethylation is a hallmark for malignant cellular transformation and could potentially be a major contributor to the oncogenic activation of HERVs .
As a result of the malignant transformation, the levels of HERV transcripts and proteins in human cancers are highly dysregulated compared with healthy tissues, where HERV expression is either low or absent . HERV-encoded protein expression in human cancer specimens is summarized in Table 2
Expression of HERV-encoded proteins detected in human cancers
|Cancer type||HERV protein||Sample type||References|
|Breast cancer||ERVK Env||Tissue|||
|ERVW Env||Cell line, tissue|||
|Endometrial carcinoma||ERVW Env||Tissue|||
|Colorectal cancer||ERVH-2 Env||Cell line|||
|Germ cell tumor||ERVK Env||Cell line||[34, 35]|
|ERVK Gag||Cell line, tissue||[35–37]|
|Melanoma||ERVK Env||Cell line, tissue||[23, 24, 39]|
|ERVK Gag||Cell line, tissue||[23, 24]|
|ERVK Rec||Cell line, tissue|||
|Ovarian cancer||ERVK Env||Cell line, tissue||[13, 40]|
|Prostate cancer||ERVK Gag||Tissue|||
|Renal cell carcinoma||ERVE-4 Env||Cell line|||
Exploiting HERV reactivation in cancer immunotherapy
+Another attractive trait of HERVs is their retroviral origin, which can promote innate immune responses and result in an adjuvant-like behavior to potentially boost antitumor immunity .
However, immunotherapy targeting HERVs is still in its infancy, but preliminary steps are being taken to elucidate the potential of HERV antigens in immunotherapy.
HERVs used as targets in monoclonal antibody therapy
ERVK Env expression has been detected at much higher levels in malignant breast cancer cell lines than in healthy breast tissues .
Wang-Johanning et al.  observed that breast cancer cell lines and human primary breast cancers have abundant levels of ERVK Env on their cell surfaces, which makes them ideal targets for antibody therapy (Figure 1).
Monoclonal ERVK Env antibodies inhibited the growth and proliferation of breast cancer cells lines in vitro.
Induction of apoptosis was also observed in breast cancer cells treated with monoclonal ERVK Env antibodies .
Mice bearing xenograft tumors showed significantly reduced growth upon treatment with monoclonal ERVK Env antibodies, compared with control experiments .
Taken together, such data indicate the potential of monoclonal ERVK Env antibodies as a novel immunotherapeutic agent for the treatment of breast cancer. Intriguingly, ERVK-positive breast tumors develop lymph node metastasis more frequently than ERVK-negative breast tumors.
Although the exact mechanism for this effect is poorly understood, it has been suggested that ERVK expression may have prognostic relevance in metastatic breast cancer . Interestingly, initial clinical studies with a humanized monoclonal antibody targeting HERV Env protein as a treatment of multiple sclerosis demonstrated a favorable safety profile with no induction of immunogenicity .
These results are promising for future clinical use of HERV-targeting monoclonal antibodies, although the potential therapeutic efficacy needs to be confirmed in more comprehensive clinical studies.Figure 1.
DNA methyltransferase inhibitors (DNMTis) alleviate the epigenetic repression of human endogenous retroviruses (HERVs) by removing DNA methylation, causing HERV double-stranded RNA (dsRNA) to activate innate immune pathway mimicking viral infections resulting in secretion of interferons (IFN).
Stimulation of interferon receptors (IFNR) activate JAK/STAT signaling and up-regulate expression of chemokines, programmed death-ligand 1 (PD-L1), cytotoxic T-cell-associated protein 4 (CTLA-4), and components of the antigen presentation pathway.
Separately, distinct HERVs exhibit tumor-associated expression similar to tumor-associated antigens (TAA) and may serve as targets for T-cell recognition, when HERV-derived peptides are presented on the cell surface in MHC class I.
Additionally, HERV envelop (Env) expression on the cell surface make it susceptible to targeting by monoclonal antibodies (mAb) or chimeric antigen receptor T cells (CART).
More information: Joel Gruchot et al, Neural Cell Responses Upon Exposure to Human Endogenous Retroviruses, Frontiers in Genetics(2019). DOI: 10.3389/fgene.2019.00655
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