WO2019201908A1

WO2019201908A1 - Method for the detection of the soluble hydrophilic oligomeric form of herv-w envelope protein

Info

Publication number: WO2019201908A1
Application number: PCT/EP2019/059788
Authority: WO
Inventors: Hervé Perron; Benjamin CHARVET; Samia KHOURY
Original assignee: Geneuro Sa
Priority date: 2018-04-17
Filing date: 2019-04-16
Publication date: 2019-10-24

Abstract

The present invention relates to an in vitro method for the detection of the soluble hydrophilic oligomeric oligomeric antigen formed by the HERV-W envelope protein in a biological fluid sample obtained from a patient suffering from an HERV-W associated disease, wherein the detection method the steps of obtaining a soluble protein fraction from said biological sample; and contacting and detecting the soluble oligomeric antigen with one or more ligand(s) targeting the SU region and/or the C terminal region of pHERV-W Envelope protein.The invention also relates to a method for the diagnosis, the therapeutic monitoring, or the prognosis of HERV-W associated diseases, or for stratifying patients suffering from an HERV-W associated disease, comprising performing the said detection method.

Description

Method for the detection of the soluble hydrophilic oligomeric form of HERV-W envelope protein

INTRODUCTION

Multiple sclerosis (MS) is an autoimmune disease characterized by multiple lesions with plaques in the brain and spinal cord. The disease is associated with an inflammatory process that attacks and destroys the myelin sheaths around axons in the brain and spinal cord. The cause of MS is unknown, although the disease is thought to be triggered by environmental factors operating on a predisposing genetic background.

MS has variable clinical presentations and highly heterogeneous disease courses, ranging from rare acute fulminate forms to benign MS without substantial disability. The great variability of this complex disease highlights the need for reliable biological markers with high sensitivity and specificity that are able to predict the future disease course and treatment response. Furthermore, stratification of MS patients with regard to their dominating pathological processes would allow individualized differential therapeutic concepts.

The search for biological markers in accessible body fluids (such as cerebrospinal fluid, CSF, blood or urine) of MS patients has been a scientific focus over the past decades. Identification of such markers would be crucial to further understanding of the etio-pathogenesis of MS, as well as for diagnosis, rational design of treatment regimens and monitoring treatment effects. Numerous biological marker candidates have been investigated so far in MS: inflammation (cytokine, chemokines, effector cell products), blood brain barrier dysfunction (adhesion molecules, matrix metalloproteases), demyelination (cytokines, antibodies, complement components, macrophage products), axonal destruction (neuronal proteins, oxidative stress, excitatory amino acids, neurofilaments, protein tau), gliosis (biochemical markers) and remyelination (myelin products, adhesion molecules, neurotrophic factors).

However, most of the obtained research results on body fluid markers in MS have been either disappointingly negative, remain controversial or still await confirmation.

Human Endogenous RetroViruses (HERVs), originating from infectious retroviruses having colonized genomic DNA through germline infections over millions of years, represent about 8% of the human genome sequences and are usually non-coding genetic elements¹. Nonetheless, several HERV copies may be expressed under physiological regulation²’³ or when activated by environmental triggers^{4 6}, thus producing ancestral retroviral proteins. Few HERV copies have been modified during evolution and are now involved in physiological functions, also called “domesticated” HERV genes, while others that retained pathogenic and/or retroviral properties are normally epigenetically repressed and remain silent, unless activated by environmental triggers following epigenetic dysregulation⁶.

Multiple Sclerosis associated Retrovirus element (MSRV), which is a member of type- W human endogenous retrovirus family (HERV-W), was first isolated from cells of patients suffering from multiple sclerosis. MSRV is normally latent in the genome of individuals, but when triggered by co-factors, such as certain common viruses, it can be reactivated and it can further express an envelope protein of the HERV-W family.

The first sequences from the HERV-W family were identified from virion-like particles produced by leptomeningeal or B-lymphocyte cultures from patients with multiple sclerosis (MS), which were then named Multiple Sclerosis associated RetroVirus⁷. HERV-W has now been shown to impact on immune cells^8,9 and on oligodendrocytes¹⁰, reproducing the hallmarks of MS pathogenesis. For instance, HERV-W pathogenic envelope protein (pHERV- W-ENV, also formerly named MSRV-ENV) promoted immune-mediated inflammation and autoimmunity in vivo, like in experimental autoimmune encephalomyelitis (EAE)¹¹ and, in vitro, impaired (re)myelination by oligodendrocyte precursor cells¹⁰.

Brain Immunohistochemistry studies, gathering over 75 multiple sclerosis (MS) cases from 7 different groups and countries, independently confirmed the presence of pHERV-W- ENV within demyelinating areas of MS lesions where it was mainly expressed by microglia, but also in a subset of lymphoid cells when present in the vicinity^{12 13}.The inventors previously investigated this mechanism and unveiled that it is a major triggering and aggravating factor in the development and progression of various diseases, collectively named HERV-W associated diseases, such as multiple sclerosis (MS), schizophrenia (SZ), bipolar disorder (BP), unipolar or psychotic depression, clinically isolated syndrome (CIS, with neurological symptom), chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, psoriasis, cancer, inflammatory pancreatitis and diabetes such as type 1 or type 2 diabetes.

Regarding MS in particular, taken together, data from successive studies suggested that an activation of HERV-W pathogenic expression may represent the missing link between environmental infectious agents and MS pathogenesis^6,14, in which the final physiopathological consequences have no validated etiological explanation to date. HERV-W elements have been detected in the serum of MS patient, however with a variable specificity and sensitivity. Indeed, MS patients whose serum and CSF samples show detectable levels of MSRV range from 50% in the French population to 100% in Sardinia. It was also detected in blood from control groups without MS (6%) (see notably Garson, J.A., et ah, Detection of virion-associated MSRV-RNA in serum of patients with multiple sclerosis. Lancet, 1998. 351(9095): p. 33, and Serra, C., et al., Multiple sclerosis and multiple sclerosis-associated retrovirus in Sardinia. Neurol Sci, 2001. 22(2): p. 171-3).

Thus there remains a need for a bio marker for the diagnosis and the prognosis of HERV-W associated diseases, notably for multiple sclerosis, which may allow with high specificity and sensitivity to identify patients, to determine the prognosis of disease progression, to predict treatment responses and to further stratify patients.

SUMMARY

The inventors have now identified a new pathogenic form of the pHERV-W Env protein in the form of a soluble hydrophilic oligomer that can be distinguished from the non- pathogenic syncytin protein.

They further provide evidence strongly supporting that such newly identified pHERV-

W Env hexamer can be considered as a hallmark of HERV-W associated diseases and in particular of multiple sclerosis (MS).

In particular, detection of the present oligomeric antigen can be used as a bio marker for early MS diagnosis (in particular before the first relapse and before MRI detection). Indeed, the results showed the detection of said hydrophilic oligomeric antigen in all MS patients that confirmed disease activity by MRI data, In clinically isolated syndromes (CIS) corresponding to a first symptomatic neurological episode, other relapse(s) or further dissemination of lesions by MRI need to be observed to diagnose definite MS versus another monophasic neurological disease. The inventors also provided evidence of the specific detection of this soluble hydrophilic pHERV-W ENV hexamer in a case of CIS that was later confirmed to be a definite MS according to previous criteria, but not in other cases without further symptomatologyThese results provide evidence that the method of the invention may be used for early diagnosis of an HERV-W associated disease, and in particular for MS.

Contrary to all expectations, the results provided by the inventors also show that this soluble hydrophilic oligomeric form of the HERV-W env protein can be found in the serum of patients. A passage of this soluble oligomeric form, from brain within the bloodstream of patients against antagonist gradients exerted by both the blood pressure and the inflammatory influx from the blood to the brain parenchyma, was indeed unlikely to occur.

Such passage within the bloodstream was even more unlikely to appear in progressive forms of MS, in which there is no, or no more, blood-brain barrier breakdown since it is normally restored. At the very contrary, the results as provided in the present application clearly demonstrate that the soluble hydrophilic oligomeric antigen is found in the serum of patients suffering from secondary progressive or primary progressive forms of MS.

It is further reminded that such high molecular weight multimeric proteins normally cannot pass blood-brain, blood-CSF and brain-CSF barriers, which is a well-known major issue for drug delivery to the brain (Pardridge 2005, Lichota, Skjorringe et al. 2010).

This newly identified oligomeric form of the HERV-W Env protein therefore represents a potent and highly specific bio marker for HERV-W associated diseases and in particular for MS, that can be easily detected from a biological fluid sample obtained from a patient suffering from an HERV-W associated disease (notably plasma, whole blood, blood cells, serum or urine), with high specificity and sensitivity.

Therefore, the present invention also relates to a method for the detection of said pHERV-W Env protein under the form of a soluble hydrophilic oligomeric antigen in a biological fluid sample.

In particular the invention relates to an in vitro method for the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W envelope protein in a biological fluid sample obtained from a patient,

wherein the detection method comprises the following steps:

obtaining a soluble hydrophilic protein fraction from said biological sample; and

- contacting and detecting the soluble oligomeric antigen with one or more ligands targeting the SU region and/or the C-terminal region of pHERV-W Envelope protein.

The present invention also relates to a method for the diagnosis, for the therapeutic monitoring, or the prognosis of an HERV-W associated disease in a patient comprising the detection of the pHERV-W Env protein under the form of a soluble hydrophilic oligomeric antigen in a biological sample, notably in a biological fluid sample obtained from said patient. The present invention also relates to a method for stratifying patients suffering from an HERV-W associated disease comprising the detection of the pHERV-W Env protein under the form of a soluble hydrophilic oligomeric antigen in a biological fluid sample obtained from said patient.

Typically, the HERV-W associated disease is selected from Multiple Sclerosis, diabetes, in particular type 1 diabetes, chronic inflammatory demyelinating polyradiculopathy (CIDP), Schizophrenia, bipolar disorder and cancer, preferably the HERV-W associated disease is multiple sclerosis (MS).

DETAILED DESCRIPTION

Definitions:

As used herein, the term "HERV-W" retrovirus, refers to the human endogenous retroviruses that comprise genetic elements (also called“copies”) belonging to the type-W endogenous retrovirus family. HERV-W is a family of human endogenous retroviruses that was unravelled in human genome from the initial discovery of“Multiple Sclerosis associated Retrovirus”, MSRV, a human retroviral element first isolated from patients with multiple sclerosis. Therefore, when studies mention“LM7” (first isolate described from MS),“MS- retrovirus”,“MSRV”,“Syncytin”,“HERV-W 7q”,“ERVW-El”,“ERVW-E2”,“HERV-W copies from X chromosome” or“HERV-W”, they all designate HERV-W elements. The terms “MRSV” or “HERV-W” both designate HERV-W elements. Specifically, the expressions "HERV-W Env" and "MSRV-Env",“pHERV-W Env (pathogenic HERV Env) all refer to the same envelope protein. MSRV-Env or pHERV-W Env is referenced under N° N°AAK181189.1 (locus AF331500 1) in Genbank. The HERV-W env protein referenced under this reference includes a signal peptide in its N terminal portion, a surface (SU) domain, a transmembrane domain and a C terminal domain (see Komurian-Pradel F et al,“ Molecular cloning and characterization of MSRV-related sequences associated with retrovirus-like particles”. Virology. 1999 Jul 20;260(l):l-9).

As used herein, the expression "HERV-W associated disease" refers to a pathological condition associated with the expression of HERV-W, preferably of the HERV-W Envelope protein. Typically, said HERV-W associated disease is a chronic inflammatory disease. As used herein, the expression " chronic inflammatory disease " refers to any disease in which persisting or recurrent inflammation is driven by innate immunity and/or by adaptive immunity involved in tissue lesions and/or can be detected locally or systemically from an overexpression of pro -inflammatory molecules. It also refers to diseases in which the inflammatory component is a pathway activating macrophagic and/or cytotoxic functions of cells from the innate immune network, including macrophages and tissue-specific macrophages and antigen-presenting cells, e.g., microglia, Kupffer or Langerhans cells, with pathogenic effects leading to targeted tissue/cellular degeneration.

Preferably, said HERV-W associated disease is selected from the group consisting of multiple sclerosis (MS), schizophrenia (SZ), bipolar disorder (BP), unipolar or psychotic depression, clinically isolated syndrome (CIS, with neurological symptom), chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, psoriasis, cancer, inflammatory pancreatitis and diabetes such as type 1 or type 2 diabetes. More preferably, said HERV-W associated disease is selected from the group consisting of Multiple Sclerosis (MS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), which both are demyelinating diseases. In one embodiment of the invention, the HERV-W associated disease is multiple sclerosis.

As used herein,“antibody” or“immunoglobulin” have the same meaning, and will be used equally in the present invention. The term“antibody” as used herein refers to immunoglobulin molecules and immuno logically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. As such, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, as well as derivatives of antibodies.

Antibodies may be human, or typically non-human antibodies such as those typically used in the field notably for diagnosis application (i.e. murine, rabbit, pig, ship, etc.).

As used herein, the expression "fragment of antibody" refers to a portion of such an antibody that mimic the hypervariable region, such as a CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3). The fragments of antibody according to the present invention retain the binding affinity and specificity of said antibody. Such fragments are functional equivalents of said antibody and they bind at substantially the same epitope as said antibody. Examples of fragments of antibody include but are not limited to heavy chain, light chain, VL, VH, Fv, Fab, Fab’, F(ab)2, and F(ab')2.

As used herein, the expression "derivative of antibody" refers to a fragment of the antibody of the invention, preferably including at least one CDR of said antibody, preferably at least one CDR3 of said antibody, fused to at least one sequence different from the natural sequence (e.g. a linker sequence of another species ...), said derivative having binding affinity and specificity to HERV-W Env comparable to that of the antibody of the invention. The derivatives according to the present invention retain the binding affinity and specificity of said antibody. Such derivatives are functional equivalents of said antibody and they bind at substantially the same epitope as said antibody. Examples of derivatives of antibody include, but are not limited to scFv, (scFv)2 and diabodies.

In natural antibodies, two heavy chains (HC) are linked to each other by disulfide bonds and each heavy chain is linked to a light chain (LC) by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Typically an antibody of the present invention is an IgG. Each chain contains distinct sequence domains.

Typically, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine the binding site specific to the antigenic epitope. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans- placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic epitope. Antibody binding sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the binding site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. As used herein, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of an antibody from any species, preferably mouse, and a CH domain and a CL domain of a human antibody.

According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of an antibody from any species, preferably mouse.

The term“Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

As used herein, The term“F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

As used herein, The term“Fab'“ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

The expressions " single chain Fv" or“scFv”" refer to a polypeptide which is a covalently linked VH::VL heterodimer, and usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

As used herein, the term“aptamer” refers to oligonucleotide or peptide molecules that bind to a specific target molecule. Nucleic acid aptamers are nucleic acid species (including DNA and RNA aptamers) that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets including proteins. Aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis and notably possess desirable storage properties. Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. Peptide aptamers can bind various cellular protein targets;

As used herein, the term "biological sample" as used herein refers to any biological sample obtained for the purpose of evaluation in vitro. In the present invention, the sample or patient sample may comprise any body fluid or tissue sample, notably tumor-specific tissues, which are typically obtained by biopsy. Preferably, the biological sample is a biological fluid, in particular in MS and diabetes. Non-limitative examples of biological fluids include whole blood (also shortly named blood), serum, blood cells, plasma, nipple aspirate fluid, urine, saliva, synovial fluid and cerebrospinal fluid (CSF). In a preferred embodiment of the present invention, the biological fluid sample is selected from whole blood, plasma, blood cells or serum. Typically the biological sample according to the invention is not a post-mortem sample.

Method for the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W envelope protein

The present application relates to the detection of an antigen consisting in the pHERV- W Env protein in the form of a soluble hydrophilic oligomer, typically a soluble hydrophilic hexamer. Said oligomeric antigen corresponds to the pathogenic form of the pHERV-W Env protein. In some embodiments, notably in MS patients, said soluble hydrophilic antigen is typically composed of monomers of the HERV-W Env protein, which signal peptide is cleaved. The present inventors have shown that it can be isolated from the soluble protein fraction of a biological sample obtained from a patient suffering from an HERV-W associated disease, most preferably from a patient suffering from multiple sclerosis. Said pHERV-W env oligomer can typically be characterized in different cumulative or alternative ways as described below:

Typically the pHERV-Env oligomer has a molecular weight comprised between 300 and 500 kDa notably between 350 and 500 kDa. More precisely, the molecular weight of the pHERV-W env oligomer is around 425 kDa (± 15 %) (i.e.: as typically detected with specific antibodies directed against epitopes of pHERV-W ENV as defined below). Said pHERV-W oligomeric antigen typically appears as a glycosylated hexamer (i.e.: composed of 6 monomers). It must be noted that both the hexameric form and apparent molecular weight should not be understood in a restrictive way, as possible variations in the number of monomers and possible truncation or cleavages of monomers may be observed and influence the global molecular weight and the composition of the oligomeric antigen of interest. Thus, said antigen composed of multiple pHERV-W ENV monomers, possibly more-or less truncated (typically with the signal peptide or not) and more-or less glycosylated, is named “oligomer” or designated as“oligomeric” in the present description. The terms“hexamer” or “hexameric” are also used in the examples but should not be intended in a restrictive way for the same reason as mentioned above.

The pathogenic antigen as herein described for detection is glycosylated. It is noted that the amount of glycosylation may be variable and thus influence the apparent molecular weight of the oligomer. Furthermore, it is to be noted that surprisingly the oligomeric structure of the antigen is typically maintained after deglycosylation or after denaturation (with or without deglycosylation). In particular, such unusual and unsuspected characteristics for a soluble hydrophilic oligomer of pHERV-W env, have notably been evidenced in body fluid samples from patients suffering from MS.

Thus the present invention relates to a method for the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W envelope protein in a biological fluid sample obtained from a patient, which comprises the following steps:

obtaining a soluble protein fraction from said biological sample; and contacting and detecting the soluble hydrophilic oligomeric antigen with one or more ligand(s) targeting the SU region and/or the C-terminal region of pHERV-W Envelope protein as previously defined. In one embodiment, detection of the said soluble hydrophilic antigen may be achieved by both one or more ligand(s) targeting the SU region and one or more ligand(s) targeting the C-terminal region (sequentially or in parallel on the same of different fractions of the biological fluid sample).

Well-suited ligands include for example aptamers or antibodies, notably monoclonal antibodies, as previously defined.

Preferred ligands, typically chosen among antibodies (typically monoclonal antibodies), are directed against an epitope localized in the N terminal region of the mature protein (i.e.: with cleaved signal peptide), which correspond to the surface SU protein domain and/or to the C terminal region (or domain) of the HERV-W Env protein.

Preferably according to the invention said ligands binding the SU region target a portion of the C terminal region of the HERV-W Env protein comprising, or consisting in, the sequence NDIEVTPP (SEQ ID N°4).

Preferably also ligands that bind to a portion of the SU domain of the HERV-W Env protein as previously defined, typically bind to a portion of the SU domain comprising, or consisting in, the sequence amino acid sequence DLYNHV (SEQ ID N°l), or to a portion of the SU domain comprising or consisting in PPMTIYTQQDLYNHVVPKPHNKG (SEQ ID N°2), or to a portion of the SU region comprising, or consisting in, PPMTI YTQQDLYNHVVPKPHKGVP (SEQ ID N°3).

Typically, said ligands are antibodies, typically monoclonal antibodies, such as monoclonal mouse antibodies. Any antibody isotype may be used according to the invention, but IgG, and notably IgGl, monoclonal antibodies are well-suited for the method of the invention as illustrated in the examples.

Preferably also, the ligands as above described detect the denatured and/or deglycosylated oligomeric (typically hexameric) form of pHERV-W ENV protein approximately within the 400-450 kDa region after separation by size of proteins extracted from the biological sample.

Suitable antibodies targeting the SU region or the C terminal region respectively include the GN mAb EnvOl (Geneuro SA, Plan-les Ouates, Switzerland), which targets the epitope DLYNHV of the SU region and the GN_mAb-Env04 (Geneuro SA, Plan-les Ouates, Switzerland), which targets the epitope NDIEVTPP of the C terminal region of the pHERV- W ENV protein monomeric sequence. It is noted that antibody GN mAb EnvOl corresponds to the antibody GNb AC1 described in the PCT application WO2010/003977. Thus typically a well-suited ligand targeting the SU region according to the present invention comprises each of the complementary-determining regions (CDRs) having the amino acid sequences SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 or any sequence having either :

- a number of substituted amino acids within said sequences as indicated in the following, and known to be feasible for obtaining functionally equivalent aminoacid sequences (Huang 1986; Zabin, Horvath et al. 1991; Edgar and Schwartz 1992; Sardana, Emini et al. 1992; Xu, Kapfer et al. 1992; Lamande and Bateman 1993; Verdoliva, Ruvo et al. 1995; Yu, Schurr et al. 1995; Wehrmann, Van Vliet et al. 1996; Ullmann, Hauswald et al. 1997; Minuth, Kramer et al. 1998; Ullmann, Hauswald et al. 2000; Janke, Martin et al. 2003): from 0 to 3 in CDR1 (SEQ ID No.l), from 0 to 2 in CDR2 (SEQ ID No. 2), from 0 to 2 in CDR3 (SEQ ID No. 3), from 0 to 1 in CDR4 (SEQ ID No. 4), From 0 to 4 in CDR5 (SEQ ID No. 5), from 0 to 2 in CDR6 (SEQ ID No. 6), or

- aminoacids substituted with other aminoacids having equivalent chemical functions and properties, as well known by the skilled man in the art (also called“aminoacid similarity”) as indicated, for an example, in the following list of similar aminoacids (one letter code): G or A, F or Y, D or E, N or Q, K or R or H, S or T, C or M, V or L or I, W or P, and/or substituted according to previous art (Huang 1986; Zabin, Horvath et al. 1991; Edgar and Schwartz 1992; Sardana, Emini et al. 1992; Xu, Kapfer et al. 1992; Lamande and Bateman 1993; Verdoliva, Ruvo et al. 1995; Yu, Schurr et al. 1995; Wehrmann, Van Vliet et al. 1996; Ullmann, Hauswald et al. 1997; Minuth, Kramer et al. 1998; Ullmann, Hauswald et al. 2000; Janke, Martin et al. 2003) within said sequences SEQ ID No.5 to SEQ ID No. 10.

These variants are the result of deletions, additions or substitutions of amino acids in the peptides of SEQ ID Nos. 5 to 10 and are also encompassed by the present invention and can be obtained by methods known in the art such as by site directed mutagenesis or by chemical synthesis.

In another aspect of the invention, said ligand, comprises a light chain variable region (VL) comprising the complementary-determining regions (CDRs) having the amino acid sequences SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7 or any sequence having at least 80% of identity and more preferably 90% of identity with said sequences, and a heavy chain variable region (VH) domain comprising the CDRs having the amino acid sequences SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 or any sequence having at least 80% of identity and more preferably 90% of identity with said sequences.

In a further aspect such ligand can comprise a light chain variable region (VL) having the amino acid sequences set forth in SEQ ID No. 11, or any sequence having at least 75% of identity and more preferably 80% and even more preferably 90% of identity with said sequence and a heavy chain variable region (VH) having the amino acid seuqence set forth in SEQ ID No. 12 or any sequence having at least 75% of identity and more preferably 80% and even more preferably 90% of identity with said sequence.

The variants of these VH and VL sequences significantly bind to the SU region of the

HERV-W env protein and notably to the epitopes of the SU region as previously mentioned.

"Sequence identity" means, for example, that in a sequence having 80% sequence identity, 80% identical amino acids are present in the same position upon alignment of the sequences, which alignment can be performed by known methods in the art such as those described in Sequence - Evolution - Function Computational Approaches in Comparative genomics. Koonon E. et al, 2003: Kluwer Academic Publishers or according to default parameters of“Mac Vector” Software (UK) instruction book.

Preferably, the ligand is a detectable ligand according to routine techniques of the field. The ligand may be labelled with a detectable label or may be detected using a secondary antibody.

Typically, monoclonal antibodies may be labelled for adapted direct detection or may be detected with secondary labelled or detectable antibodies according to routine immunodetection techniques. Obtainment of a soluble protein fraction from a biological sample can be achieved according to routine techniques in biochemistry including extraction of the total protein fraction from the biological sample and separation of the soluble hydrophilic proteins from the total protein fraction. Optionally, the proteins from the total protein fraction or from the soluble fraction can be denatured. Typically extraction of the total protein fraction is performed in denaturing conditions according to routine techniques in the field (for example as illustrated in the results by using a buffer containing a denaturing agent (typically ionic detergents, notably comprising polar detergents) optionally with a reducing agent, and by heating the denaturing medium containing the proteins at a temperature between 70 and l00°C).

Preferably the step of contacting and detecting the soluble hydrophilic oligomeric antigen with one or more ligand(s), as defined above, is performed on proteins of more than 100, more than 150, more than 200, more than 250, more than 300, more than 350 kDa, or more than 400 kDa. Thus, the method of the invention may further comprise a step of size separation of the proteins, which can be performed on the total protein fraction, or preferably on the soluble protein fraction, obtained from the biological sample. More generally a potential step of size separation of protein of more than 100 kDa as defined above is typically performed before the step of contacting and detecting the soluble hydrophilic oligomeric antigen with a suitable ligand as described above. Preferably the size separation discriminates proteins of high molecular weight notably proteins above 100, 150, 200, 250, 300 or preferably above 350 kDa.

The inventors have further shown that detection of the soluble hydrophilic oligomeric pHERV-W (typically in the form of an hexamer and as previously defined) from a biological sample obtained from a patient suffering from a HERV-W associated disease (typically a MS patient), with a ligand targeting the C terminal region of the HERV-W env protein monomers can only be achieved after deglycosylation (typically performed after protein denaturation) of the said antigen. In particular the results provided herein show that the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W envelope protein by a ligand targeting the C terminal region of the HERV-W env protein (notably the amino acid sequence NDIEVTPP) is a hallmark of the pathogenic form of HERV-W envelope protein, in particular in active brain lesions from MS.

Thus in one embodiment, the method of the invention includes a step of protein deglycosylation before contacting the proteins with a suitable ligand as described above. Thus the deglycosylation step is performed on the total protein fraction or preferably on the soluble protein fraction. Most preferably, the step of protein deglycosylation is performed on denaturated protein. The deglycosylation step can be performed before or after any step of size separation of the proteins. Deglycosylation may be achieved by any protocol known by the one skilled in the art, using typically “deglycosylation kits” which are currently commercially available.

In one embodiment of the method of the invention, the biological sample obtained from the patient suffering from a HERV-W associated disease is fractioned and the sample fractions are submitted to detection:

- a ligand (preferably an antibody) targeting the SU region as above defined, and

(typically in parallel)

- a ligand (preferably an antibody) targeting the C terminal region as above defined.

Typically as detailed above, the step of soluble protein extraction (with protein denaturation or not) and potentially the the step of protein deglycosylation and/or the step of protein size separation (in order to discriminate protein of high molecular weight as previously defined) are performed before detection with the ligands (notably before immune- detection).

In one embodiment, the sample is size- fractioned during electrophoretic migration and each size-fraction (but preferably the size fraction(s) corresponding to the expected molecular size of the oligomeric pHERV-W antigen as previously defined) of the sample is submitted to immunodetection with one or more antibody(ies) as detailed above (in parallel or not).

In one embodiment, immuno-detection with an antibody targeting the SU region may be performed on a biological sample as defined above before, preferably after a step of deglycosylation of the protein. Preferably the biological sample is fractioned before the I^st immune detection and may be submitted to any further immune detection with the same antibody or with another antibody targeting the C-terminal region.

In various embodiments, the in vitro method for the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W env protein in a biological fluid sample obtained from a patient comprises the following steps:

A: extracting the total protein fraction from the said biological sample, obtaining a protein soluble hydrophilic fraction from the total protein fraction, and immunodetecting the soluble hydrophilic oligomeric antigen with an antibody targeting an epitope comprising or defined by the amino acid sequence DLYNHV; wherein the proteins from the total protein fraction or from the soluble protein fraction are optionally denaturated and preferably further deglycosylated before the immunodetection of the soluble hydrophilic oligomeric antigen;

either alternatively, or as a confirmation method,:

B.

extracting the total protein fraction from the said biological sample,

- obtaining a protein soluble hydrophilic fraction from the total protein fraction

immunodetecting the soluble hydrophilic oligomeric antigen with an antibody targeting an epitope located in the C terminal region of the pHERV-W protein monomer, and notably comprising the sequence NDIEVTPP;

wherein the proteins are optionally denaturated and preferably further deglycosylated before the step of immunodetection of the soluble hydrophilic oligomeric antigen.

The biological fluid sample is preferably size-fractioned at any step before immune-detection and submitted to parallel immune detection with both an antibody targeting the SU region and an antibody targeting the C terminal region. Such embodiment may be typically used for the detection of the soluble hydrophilic oligomeric antigen from a biological sample obtained from a patient suffering from MS, in particular for the confirmation of MS disease activity.

Size separation (or size- fractioning) and immunodetection of the oligomeric antigen as herein defined may be achieved typically using capillary electrophoretic migration of the protein from the soluble fraction obtained from the biological sample, with one or more antibody targeting the SU region and/or the C terminal region as previously defined. When an antibody targeting the C terminal region is used, a step of deglycosylation of the protein is typically performed before immunodetection. Typically, the immunodetection is performed for proteins that migrate in a region corresponding to an apparent molecular weight ranging from from 300 to 500 kDa, notably from 350 to 500 kDa, notably from 400 to 450 kDa.

In one embodiment the soluble hydrophilic oligomeric antigen as herein defined may be detected and further quantified in the biological sample. In some embodiments of the method of the invention the level of the soluble hydrophilic pHERV-W oligomeric antigen may be compared, and notably normalized with respect to a control level or value of a reference protein or of a technical marker. A control value may also correspond to a mean value obtained from control biological sample, to determine a technical threshold that allows to statistically significantly discriminate negative samples from positive ones. Control biological sample may be obtained from one or more control healthy subjects, (typically subjects which are not suffering from an HERV-W associated disease).

In vitro methods for the diagnosis, the therapeutic monitoring or the prognosis of

HERV-W envelope protein associated disease and for the stratification of patients suffering from an HERV-W associated disease

The present invention also encompasses an in vitro method for the diagnosis and/or the therapeutic monitoring, and/or the prognosis of an HERV-W envelope protein associated disease in a patient.

Said method comprises performing the detection method as previously detailed.

Preferably the HERV-W envelope protein associated disease is multiple sclerosis (MS). As previously indicated the authors have shown that the soluble hydrophilic antigen can be detected at an early stage of the disease. Thus the method of the invention allows establishing an early diagnostic of the disease such that an appropriate treatment strategy may be proposed.

Typically according to the invention, a patient is suffering from an HERV-W env protein associated disease, notably multiple sclerosis, or is at risk of suffering of such a disease. In one embodiment, the patient is at risk of suffering of MS. The patient is typically a human.

Typically when the patient is suffering from multiple sclerosis, the method allows to predict or diagnose active phases of the disease such that the treatment may be early adapted.

The method of the invention also allows stratification of the patients suffering from an HERV-W associated disease, notably of the MS patients.

In one embodiment, the soluble hydrophilic oligomeric antigen form by the HERV-W env protein as herein defined is further quantified in the biological sample. Detection and quantification of such pathogenic antigen may also allow to establish a prognosis regarding the evolution of the disease and/or to predict a response to treatment, or to stratify patients with regards the severity of the disease or to the stage of the disease.

The present method may also be performed at various time points for the follow up of the disease, the levels of the soluble hydrophilic oligomeric antigen may thus be compared (or normalized) with respect to a control value (as above defined) or with respect to prior value obtained in the same patient. In such context the method of the invention is particularly advantageous as it can be easily performed on a biological fluid sample from the patient while preserving both high specificity and sensitivity. The method of the invention may allow to refine prognosis of the disease and/or to precisely adapt the treatment. For example the method may allow to predict MS relapse and to provide an adapted treatment.

The present invention also encompasses the use of an antigen consisting in the soluble hydrophilic oligomeric pathogenic antigen formed by the HERV-W envelope protein, as herein defined as a biomarker for the diagnosis of and HERV-W env protein associated disease.

Kit of the present invention

The present invention also encompasses a kit adapted to perform the method of detection or the method for the diagnosis, the therapeutic monitoring or the prognosis of HERV-W envelope protein associated disease, or the method for the stratification of patients suffering from an HERV-W associated disease, as herein described.

Typically such kit comprises: an antibody targeting the N-terminal region, notably the SU domain of the pHERV-W env protein as defined previously; and

an antibody targeting the C-terminal region of the pHERV-W env protein as previously defined.

As a matter of illustration, a well-suited kit according to the invention comprises the GN_mAb-ENV0l and the GN_mAb-ENV04 antibodies. Method of treatment of an HERV-W envelope protein associated disease

The present invention also encompasses a method of treating and HERV-W envelope protein associated disease comprising:

performing the diagnostic or the detection method as previously described, and - administering an appropriate treatment.

An appropriate treatment as per the invention encompasses any adapted prophylactic or curative treatments. Suitable appropriate treatment typically includes antibodies as described herein as well as in applications W02010/003977 and WO2015/181226, and notably the HERV-W Env 001 antibody (corresponding to the GNbACl antibody). A therapeutic antibody, as per the therapeutic method of the invention, can be a human antibody or a chimeric antibody including human constants domains, or a humanized antibody. A pharmaceutical composition comprising GN mAb-ENVOl antibody, or an antibody as described in WO2010/003977 and WO2015/181226 or a chimeric or humanized version of such antibodies can typically be administered in amounts that will be therapeutically effective and immunogenic and as known in the art, the dosage that is administered depends on the individual to be treated.

FIGURES

Figure 1: Reference proteins and antibodies:

Detection of recombinant pHERV-W ENV proteins by capillary immunoelectrophoresis

A: E-coli recombinant pHERV-W ENV protein comprising the signal peptide and glycosylated recombinant protein from transfected human cells (HEK) with cleaved signal peptide. The localization of epitopes targeted by specific antibodies is illustrated on the E. coli protein schematic representation.

B: Chromatographic and Western-blot immunodetection of these two recombinant proteins with GN mAb-EnvOl monoclonal antibody. The theoretical molecular weights calculated from amino acid sequence, 62kDa of pHERV-W ENV produced in E. coli and 58kDa when produced with cleaved signal peptide in mammalian cells, were confirmed. Differences in size are mostly due to the cleavage of the signal peptide, which is not compensated by glycosylations. Figure 2: Biochemical forms and sequence analysis of pHERV-W-ENV antigen

Immunoelectrophoretic profiles of HERV-W proteins expressed in transfected human cells under denaturing conditions with GN mAb-ENVOl (A-B) or GN_mAb-ENV04 (C-D).

A-D monomeric and oligomeric profiles represented with Western blot (top) and chromatographic profiles (bottom).

A&C: Capillary detection of molecular weights ranging from 12 to 230 KDa (optimum: 40- 200 KDa)

B&D: Capillary detection of molecular weights ranging from 66 to 440 Kda (optimum: 200- 450 KDa)

Top lane: Mock transfected HEK cells with same plasmid encoding GFP protein.

Middle Lane: Proteins extracted from HEK cells transfected with original MS-derived clone (MSRV pVl4; GenBank no. AF331500.1) representing the pathogenic HERV-W ENV protein, pHERV-W ENV

Bottom Lane: Proteins extracted from HEK cells transfected with human placenta-derived syncytin clone representing the physiologically“adopted” HERV-W ENV protein involved in placental syncytio-trophoblast cell fusion, Syncytin (pH74; GenBank no. AF072506.2).

E: Aligned aminoacid sequences of pHERV-W ENV (formerly MSRV-ENV; upper line; SEQ ID N°l3) and Syncytin-l (formerly HERV-W/7q-ENV; bottom line). In the middle line, when aminoacids are identical, their one-letter code is repeated, when substituted by an aminoacid with analogous chemical properties it is indicated by“+”, the absence of letter or a black rectangle (thus highlighted in critical regions), indicates non-homologous substitution with aminoacids displaying divergent properties. The epitope specifically recognized by GN mAb- ENV01 is highlighted in grey on line 301 to 360. The epitope specifically recognized by GN_mAb-ENV04 is highlighted in grey on line 481 to 540. The black box delineates the sequence corresponding to site of trimerization of retroviral envelope glycoproteins. Inside the back box, the grey 38 box delineates the sequence corresponding to site of cleavage by Furin enzyme in retroviral envelope glycoproteins

F: Evidence that pHERV-W-ENV dimer was generated by denaturation. No dimer was observed in samples denatured after column filtration (association with large partner, retained by filter). Dimer was observed in samples denatured before column filtration (association dimer-partner was broken and isolated dimer can be eluted through the filter) Figure 3: Water solubility of pHERV-W-ENV antigens

Immunoelectrophoretic profiles of HERV-W proteins expressed in transfected human cells under denaturing conditions with GN mAb-ENVOl. Protein extraction from HEK cells transfected with original MS-derived clone (MSRV pVl4; GenBank no. AF331500.1) representing the pathogenic HERV-W ENV protein, pHERV-W ENV. Monomeric and oligomeric profiles represented with Western blot (top) and chromatographic profiles (bottom).

Left: Capillary detection of molecular weights ranging from 12 to 230 KDa (optimum: 40-200 KDa)

Right: Capillary detection of molecular weights ranging from 66 to 440 Kda (optimum: 200- 450 KDa)

Capillary detection of protein extracted from HEK cells transfected with original MS-derived clone (MSRV pVl4; GenBank no. AF331500.1) representing the pathogenic HERV-W ENV protein, pHERV-W ENV.

Soluble fraction (hydrophylic): Western blot top lane and chromatogram as indicated.

Non-soluble fraction (hydrophobic): Western blot bottom lane and chromatogram as indicated.

Figure 4: pHERV-W-ENV oligomers from transfected cells dissociate after de- glycosylation

Left: Capillary detection of molecular weights ranging from 12 to 230 KDa (optimum: 40-200 KDa) Right: Capillary detection of molecular weights ranging from 66 to 440 Kda (optimum: 200- 450 KDa)

Non-deglycosylated proteins: Western blot top lane and chromatogram as indicated.

Deglycosylated proteins : Western blot bottom lane and red chromatogram as indicated.

Figure 5: pHERV-W-ENV soluble and stable hexamer detection is a hallmark of MS active lesions

Immunoelectrophoretic profiles of HERV-W proteins detected from frozen brain white matter blocks, after protein extraction and separation of proteins with KDa>200 KDa in the soluble fraction. Analysis is performed under denaturing conditions with GN mAb-ENVOl (A-D) or GN_mAb-EN V 04 (E-H).

A-D: Non-deglycosylated soluble protein fractions (MW>200 Kda) with with GN mAb- ENV01.

A: Samples from MS active lesions. Detection of hexameric oligomer in the soluble fraction of all samples between 400-450 KDa. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

B: Samples from normal appearing white matter (NAWM) of MS brains. Detection of hexameric oligomer in only one sample between 400-450 KDa. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

C: Samples from white matter (NAWM) of brains from non-MS controls. Detection of hexameric oligomer in only one sample with brain metastasis of prostate cancer. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

D: Graphical representation of the calculated area under the curve (AUC) of individual chromatograms from all samples. From left to right : non MS controls, NAWMs from MS and MS lesions. *: p<0,05; **: p<0,0l.

E-G: Deglycosylated soluble protein fractions (MW>200 Kda) with GN_mAb-ENV04.

E: Samples from MS active lesions. Detection of hexameric oligomer in the soluble fraction of all samples between 400-450 KDa. Detection is presented with Western blot (top) and chromatographic profiles (bottom). F: Samples from normal appearing white matter (NAWM) of MS brains. No more detection is seen between 400-450 KDa in MS NAWM samples. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

G: Samples from white matter of brains from non-MS controls. No more detection is seen between 400-450 KDa in MS NAWM samples. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

H: Graphical representation of the calculated area under the curve (AUC) of individual chromatograms from all samples. From left to right : non MS controls, NAWMs from MS and MS lesions. ***: p<0,00l; ****: p<0,000l.

I-K: Non-deglycosylated soluble protein fractions (MW>200 Kda) with GN_mAb-ENV04.

I: Samples from MS active lesions. No detection of hexameric oligomer in the soluble fraction of all samples between 400-450 Kda (box with dotted line). Detection is presented with Western blot (top) and chromatographic profiles (bottom).

J: Samples from normal appearing white matter (NAWM) of MS brains. No detection is seen between 400-450 KDa (red box with dotted line) in MS NAWM samples, but a non-specific background is observed from 200KDa in the majority of MS NAWM samples. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

K: Samples from white matter of brains from non-MS controls. No detection is seen between 400-450 KDa (box with dotted line) in control brain samples. A non-specific background is also observed from 200KDa in one sample. Detection is presented with Western blot (top) and chromatographic profiles (bottom).

Figure 6: Macromolecular model for pHERV-W-ENV antigen forms from MS brain lesions.

A: Non-deglycosylated proteins. The monomeric, trimeric and hexameric forms are schematically represented and the binding of specific monoclonal antibodies targeting the surface/hydrophylic domain or the“transmembrane’Vhydrophobic domain is also shown.

B: Deglycosylated hexamer. Epitope accessibility for GN_mAb_Env04 to internalized hydrophobic domain with c-term extremity was not possible with glycosylations in the hexameric form, but becomes possible after deglycosylation that unfolds the structure. Deglycosylation does not disrupt the hexameric association from active brain lesions, unlike the hexamer obtained from in vitro transfected cells.

Figure 7: pHERV-W-ENV soluble hexameric antigen detected in MS sera

Detection of non-deglycosylated soluble pHERV-W ENV hexamer protein by capillary immuno electrophoresis with GN mAb-ENVOl in sera from MS and from healthy controls, after protein extraction and separation of proteins with KDa>l50 KDa. Graphical representation of the calculated area under the curve (AUC) of individual chromatograms from all samples (A-B).

A: MS sera versus Healthy blood donors (HBD).

B: HBD samples are compared to different forms of MS: Primary Progressive MS-PPMS, Relapsing-Remitting MS, and Secondary Progressive MS (SPMS). Clinically Isolated Syndromes (CIS, cases with initial symptoms awaiting confirmation of an MS diagnosis according to clinical evolution and/omew lesions detected by magnetic resonance imaging- MRI).

P values showing statistical significance are indicated above the corresponding bars between compared groups.

EXAMPLES:

MATERIALS AND METHODS

Purified recombinant full-length protein produced in E-coli:

HEK293T transfected cells: HEK293T cells (ATCC® CRL-3216™) were transfected with three different constructs: pMAX-GFP, encoding for GFP and used as control, pCMV- MSRV-ENV encompassing complete orf of pHERV-W ENV (MSRV-ENV clone, 542 amino acids GenBank no. AF331500.1) or pCMV-SYNl (cDNA from full-length placenta RNA encoding Syncytin-l, 538 aminoacids-GenBank no. AF072506.2) under CMV promoter control (Geneuro, Switzerland). Transfected cells were harvested 24h post transfection

Brain samples: Snap frozen necropsy brain samples: 5 from active MS lesions, 4 from MS normal appearing white matter (NAWM) and 4 from non-MS controls. Sera from MS and controls: Sera from Multiple Sclerosis (MS) patients with remitting- relapsing, secondary progressive or primary progressive forms (respectively, RRMS, SPMS and PPMS), from Clinically Isolated -neurological- Syndrome (CIS), and from healthy controls were obtained in the same conditions.

Protein extraction: Cell pellets or supernatants were placed in extraction buffer, containing RIPA buffer (Sigma Aldrich) supplemented with 1% Fos cholin (Anatrace) and protease inhibitor cocktail (Roche). Total protein mixture was then lysed using a crusher during 4 successive runs of lOs, velocity 4, on ice. Lysates were incubated for 2h at 25°C with gentle agitation (120 rpm). Thus, extracts from cell pellets were centrifuged lOmin at 10 OOOg. Supernatants were aliquoted and named“soluble fraction” and pellets were resuspended in extraction buffer, aliquoted, and named “Insoluble fraction”. Lysates extracted from supernatants were not centrifuged and were used as“total fraction”. Total protein amount from each fractions were evaluated using the“Protein Assay Reagent” kit (Pierce).

Protein size separation using AMICON columns: 500pL of protein extract were load in “AMICON Ultra-0.5 100K” device (Merck-Millipore). High molecular weight proteins (>l00kDa) were purified / concentrated on column filter by centrifugation 30min, 14 000 x g. Filtrate (proteins <l00kDa, 450pL) and concentrate (>l00kDa, 50pL) were independently conserved at -80°C.

Deglycosylation: 50pg of total protein extract were deglycosylated using “Protein Deglycosylation Kit” (Promega). Deglycosylation was performed according manufacturer instructions during 18h at 37°C.

Automated capillary western blot: Western blots were performed using WES, an automated capillary-based size sorting system (Proteinsimple). All procedures were performed with manufacturer's reagents according to their user manual. Briefly, diluted protein lysate was mixed with fluorescent master mix and heated at 95°C for 5 minutes. The samples (2mg/mL for untreated extracts or 300pg/mL after deglycosylation), blocking reagent, wash buffer, primary antibodies, streptavidin or secondary antibodies coupled with HRP, and chemiluminescent substrate were dispensed into designated wells in a manufacturer provided microplate. The plate was loaded into the instrument and protein was drawn into individual capillaries on a 25 capillary cassette (66-440kDa) provided by the manufacturer. Protein separation and immunodetection was performed automatically on the individual capillaries using default settings. Primary antibodies, GN mAb EnvOl -biotin (20mg/mL) or GN_mAb_Env04 (35mg/mL) were raised against pHERV-W ENV protein produced by clones from MS retroviral particles¹².

The secondary antibody used for the luminometric detection with Env-04 is an anti-mouse secondary antibody from ProteinSimple, USA (cat. # 042-205) as part of their kit“Anti- Mouse Detection Module ProteinSimple DM-002 ». A schematic representation of HERV-W Envelope regions of epitopes targeted by specific monoclonals is represented in Figure 1E, along with Aligned MSRV and Syncytin-l sequences.

Globally, the theoretical hexamer molecular weight is around 360 kDa, therefore expected in- between 300 and 500 kDa, depending on the conditions applied to the migration and to the separation by size of the proteins containing the target hexamer of interest. In the examples providing the evidence of the detection of the present invention, a capillary immuno electrophoresis system has been used with its associated software (WES platform, Proteinsimple, USA). Using this WES system, the apparent molecular weight at which the peak of luminometric detection corresponds to the detection of the pHERV-W ENV hexamer- like oligomeric form in the present experimental conditions ranges approximately between 400 and 450kDa. This does not preclude variations between experiments on the same WES device, between different WES devices and with experiments performed on other platforms, in particular with alternative technical settings and principles for separating proteins from a sample and determining their apparent molecular weights. Typically, the term“hexameric” hydrophilic soluble pHERV-W antigen designates the protein oligomer composed of multiple pHERV-W ENV monomers, associated or not with other unrelated molecules, which is detected in a region corresponding to an apparent molecular weight between 300 and 500 kDa and therefore appears to correspond to an hexameric oligomer (6 monomers), in particular with glycosylations that can be variable, but is not restrictive to an oligomer contain 6 monomers of pHERV-W protein.

The Area under the curve (AUC) of interest 400-450 kDa as observed in the conditions of the present examples of the invention, were calculated by the Compass software (ProteinSimple) and all values were computed on Graph Pad 7 software (PRISM). This allows, for instance, a quantification of the oligomer of interest by obtaining the AUC value between 400 and 450 kDa, between values delimiting the corresponding peak. A quantification of the corresponding mass of protein can be obtained from known quantities of a protein standard that can be spiked into the medium before loading the system of immunoanalysis or, in parallel with the same platform or device, by determining a calibration curve plotted from AUC values in abscises and from corresponding mass values (mg, pg, ng or pg, etc.) in ordinates, as commonly used by persons skilled in the art. Alternatively, an AUC value can be obtained from an irrelevant but constantly present peak (Internal Standard) in another region of detection (other kDa) from the same types of samples, when analyzed in the same conditions, and a ratio can be calculated between the AUC of the peak for the oligomer of interest and the AUC for the Internal Standard. The latter ratio thus allows to normalize the inter assay variations, if occurring, and therefore allows comparing results from different samples of the same or of different patients for appropriate evaluations and studies. Moreover, such calculated values, presented as a mass of protein deduced from a standard curve and/or a ratio with an Internal Standard, allow to define a statistical threshold (cut-off) above which values can be confirmed to be positive for the presence of the pHERV-W oligomer of interest, when compared to normal healthy control samples in which it is not present. This can be calculated from residual AUC values, consisting in background and non-specific signal generated by the technique and the sample type (background noise) by the same software(s) in the same analytical conditions of a panel of similar samples from healthy individuals. The usual calculation consists in determining the mean (M) and standard deviation (SD) values of the“healthy” samples, given that outliers with aberrant signal of unknown origin are excluded if ever observed. The, the cut-off can be calculated as M+2SD (as recommended in epidemiological studies: http://www.who.int/nutgrowthdb/about/introduction/en/index5.html) or, preferentially, as M+3SD (commonly taken for 99% confidence interval), or M+ 3.08 SD. Thus, values above the cut-off correspond to positive samples containing the oligomer of interest and values below the cut-off correspond to negative values of samples devoid of this oligomer or with quantities below the limit of detection of the technique that has been used. The determination of the limit of detection of a given technique and protocol is a well-known procedure that can be applied to protein quantification by a person skilled in the art.

AUC values, ratios or other result values presented on graphic representations in figures referred to in the following examples, were expressed as the mean ± SEM (Standard Error of the mean). RESULTS

1. Immunodetection profiles of recombinant HERV-W envelope proteins

As illustrated in Figure 1A, an HERV-W ENV monomer from MSRV clone was produced and purified from E. coli as a non-cleaved full-length protein, retaining the signal peptide and intra-cellular tail. The same cloned cDNA from RNA of purified retrovirus- like particles produced by cultured cells from a patient with MS (pVl4; GenBank no. AF331500.1), was inserted in a plasmid suitable for transfection in human cells with cytomegalovirus (CMV) promoter.

As represented in Figure 1 A, the expressed pHERV-W ENV protein (formerly MSRV

ENV), after transfection of human embryonal kidney cells (HEK 295-T) with this plasmid, is glycosylated with cleaved signal peptide. This results in a difference in the apparent molecular weight (MW) of the two monomeric proteins, as detected by capillary immuno electrophoresis (Figure 1B).

Syncytin-l (SYN-l; formerly HERV-W/7q ENV) is another envelope protein from

HERV-W family, which has acquired a unique physiologic function during evolution and now mediates cell-fusion restricted in time and space to the formation of placenta syncytiotrophoblast tissue. As shown with alignment of their aminoacid sequences in Figure 1B, pHERV-W ENV and SYN-l share 87% of identical aminoacids, 90% when including chemically analogous aminoacids, but SYN-l shows a gap of 4 aminoacids in the C-terminal region that correspond to 12 nucleotide deletion in the corresponding DNA sequence. This reveals to be a molecular signature for SYN-l since, to date, all other identified HERV-W copy with env gene revealed homologous to coding sequence of pHERVW ENV, i.e. they do not lack these 12 nucleotides. Thus, the cDNA from full-length placenta RNA encoding Syncytin-l (pH74; GenBank no. AF072506.2) was sub-cloned in the same expression pCMV plasmid suitable for transfection of HEK cells, in parallel with pVl4 and with a control protein-coding insert within the same plasmid (GFP).

In Figure 2A, equal quantities of extracted proteins from HEK-transfected cells showing similar expression efficacy with the three plasmids have been analyzed in parallel by capillary Immunoelectrophoresis with two different monoclonal antibodies raised against pHERV-W ENV protein, which target regions indicated in Figure 1A and specific epitopes highlighted in Figure 2B. Whereas results do not show specific detection of SYN-l and GFP control proteins with both antibodies, they clearly reveal that pHERV-W ENV is well detected by both antibodies. Importantly, it also clearly show that its expression generates an oligomeric pattern with, in addition to the monomeric glycosylated form with cleaved propeptide detected around 58 KDa, trimeric and hexameric forms respectively detected around 185 KDa and 440 KDa. These three forms are detected under non-denaturing conditions (not shown) and, under reducing and denaturing conditions, a significant proportion of dimer and a lower proportion of tetramer are generated and can also be detected on chromatograms and western blot analyses (Figure 2A).

Nonetheless, the furin cleavage site between SU and TM regions in pHERV-W ENV appears not to be functional with the observed aminoacid sequences identified in this region. This is shown by the size of the detected monomers and oligomers that are detected by both monoclonal antibodies targeting either the SU or the TM domain in Figure 1B. Interestingly, the region of the furin cleavage site appears to have remained canonical in SYN-l sequence (Figure 2B) and, conversely, the sequence region of the trimerization site of retroviral envelope glycoproteins has not been conserved in SYN-l sequence whereas it has remained canonical in pHERV-W ENV. This should explain the observed oligomeric pattern of pHERV-W antigens detected by the present analyses.

2. Solubility, hydrophilicity and hydrophobicity of pHERV-W ENV antigen forms

Because these different oligomers may confer diverging properties to these macro molecular structures, we have analyzed their solubility in aqueous buffers used for protein extraction.

As shown in Figure 3, nearly all the hexamer was detected in the soluble fraction, thus revealing its global hydrophilicity, whereas the monomeric and trimeric forms were conversely detected in the insoluble (hydrophobic) fraction. The dimeric form, though dominantly detected in the insoluble fraction, was also detected in the soluble one. However, as resulting from experimental denaturation, this may not have biological significance.

Therefore, the pHERV-W ENV hexamer represents the only soluble (hydrophilic) physiological form of corresponding envelope protein antigens. 3. Influence of glvcosylations

Because dealing with a retroviral envelope glycoprotein, though of endogenous origin, glycosylations are expected on canonical sites as illustrated in Figure 1A. These may be variable according to the expressing cell but, as a reference, pHERV-W ENV from HEK- transfected cells was analyzed by capillary immunoelectrophoresis comparing the same quantity of extracted protein with and without enzymatic deglycosylation. Results presented in figure 4 clearly show that deglycosylation nearly disrupted all hexameric macro molecules from the sample, and relatively had the same effect on the trimer. Interestingly, this was accompanied by a strong increase of the WB monomeric band and by the corresponding area under the curve seen on the chromatogram. The non-physio logical dimeric form was apparently not affected. These results show that glycosylation of the pHERV-W ENV monomers in trimeric and hexameric forms play an important role, at least in the stability of these oligomeric forms. Here, with glycoproteins from HEK-transfected cells, the effect of deglycosylation essentially appears to generate monomers.

4. Soluble high molecular weight pHERV-W ENV in MS brain lesions.

pHERV-W ENV (then named from its former denomination MSRV-ENV) was detected in all MS post-mortem brains analyzed to date, quite selectively associated with areas of still ongoing demyelination in necropsic tissue¹³. Moreover, producing cells appeared to be dominantly microglia and non-CD3 cells of lymphoid cuffs in the vicinity of actively demyelinating area. Therefore, in order to determine the molecular profile and the solubility of HERV-W ENV antigen in MS brains, we have analyzed frozen tissue blocks from MS active lesions, from MS normal appearing white matter (NAWM) and from white matter (WM) of non-MS brains, with the same capillary immuno electrophoretic approach as previously for proteins from reference clones. In preliminary analyses of three MS brain samples, the total protein extracts (not separated by water solubility) from active MS lesions showed detectable HERV-ENV monomer, trimer and hexamer, bur not NAWM (data not shown). However, more specifically addressing the question of a soluble macromolecule, we observed the results presented in Figure 5, with high molecular weight proteins from the soluble fraction previously separated by size exclusion (Cf. Materials and Methods).

These results show, firstly, a soluble antigen with the apparent MW of pHERV-W ENV hexamer and the specific detection with GN_mAb-ENVOl (specific for the surface domain-SU, Cf. Fig.1) in all samples from MS active lesions. This was detected only from one tissue block of NAWM and, in non-MS WM, only a sample from a brain with metastases of prostate carcinoma showed a similar signal. Nonetheless, as represented in Figure 5D, the calculated area under the curve (AUC) from chromatograms between 400 and 450 KDa for each sample already showed a significant difference between MS active lesions and MS NAWM, still emphasized between MS lesions and non-MS WM.

Secondly, deglycosylation of the same soluble protein extract showed a stable detection of HERV-W Env antigen with GN_mAb-ENV04 antibody specific for the C- terminal“tail” of its transmembrane domain (TM; Cf. FiglA) in all MS active lesions. It therefore confirmed the presence of the entire structure of the entire pHERV-W protein in a macro molecule corresponding to its hexameric form as previously defined. Most importantly, this oligomerization and the presence of a complete SU-TM protein without furin cleavage of the subunits confirmed by two monoclonal antibodies with distant epitopes not detected in Syncytin-l with the same analysis, now evidences that the HERV-W antigen expressed in MS brain lesions is pHERV-W (MSRV) envelope glycoprotein and not its sister protein Syncytin- 1. Conversely, the macro molecular structure detected at the same MW in MS NAWM and in

WM with metastases was no longer detectable at this MW similarly to what was previously observed after deglycosylation of trimeric and hexameric forms of pHERV-W ENV produced by transfection in HEK cells. This also indicates that, in addition to the fact that pHERV-W ENV soluble hexamer is a hallmark of MS demyelinating lesions, it also has unique properties that maintain its oligomeric structure after deglycosylation.

In figure 5, the histograms representing the AUC of deglycosylated pHERV-W soluble hexamer with GN_mAb-ENV04, also showed the unique specificity of this detection when quantified in active MS lesions versus all samples from MS NAWM and non-MS WM.

The molecular analysis also provided another unique and interesting feature, as shown in Figure 5 I-K. In these analyses made with GN_mAb-ENV04 antibody, all samples from MS and non-MS brains could not detect the pHERV-W epitope in the non-deglycosylated form corresponding to the hexameric form. Only more-or-less intense non-specific background signal was observed in few samples, but not at the specific MW around 440 KDa. This indicated that the C-terminal epitope was not accessible to its specific antibody in glycosylated hexameric forms of pHERV-W expressed in the brain, conversely to the oligomer from HEK transfected cells. It also evidenced that, though not disrupting the oligomeric structure of pHERV-W soluble antigen from MS active lesions, deglycosylation modified its macromolecular structure in a way that made TM regions accessible to antibodies and exposed the epitope specifically recognized by GN_mAb-ENV04 (Cf. Fig. 1 A-B).

Taking into account all these newly obtained data on pHERV-W ENV macromolecular characteristics when expressed in different tissues or cells, a global structural model is proposed in Figure 6 that explains them altogether.

5. pHERV-W ENV soluble hexameric macromolecule is detected in sera from patients with MS

After having evidenced the specific macromolecular characteristics of the pHERV-W ENV antigen expressed in MS brain tissue with active demyelination, a passage of this soluble hexameric form within the bloodstream of MS patients against antagonist gradients exerted by the blood pressure and by the inflammatory influx from the blood to the brain parenchyma was unlikely to occur.

We nonetheless analyzed sera from active (with clinical exacerbation and/or MRI new lesions) MS patients, versus healthy controls obtained under the same conditions in the same population.

Sera were directly used to extract proteins, separate the soluble fraction, then the high molecular weight (>150 KDa) molecules. Results from capillary immuno electrophoresis with GN_mAb-ENV0l antibody on non-deglycosylated proteins are presented in figure 7, with individual AUC of chromatograms showing unexpected immunodetection within the 400-450 KDa region, as plotted on the graph.

In these conditions, the soluble oligomer was readily detected in nearly all active MS patients (80%), and in 100% of patients with Gadolinium-enhanced Tl MRI detected lesions in brain. The group of healthy controls showed homogeneous negative results with similar background signal, with a very significant difference between the two groups (p<0.0004). Interestingly, all MS patients had values above controls. When analyzing different sub-groups with different clinical forms or phases of the disease as shown in Figure 7B, it appeared that detection was possible even in clinically isolated syndromes (CIS) corresponding to a first symptomatic episode still requiring another relapse or the dissemination of lesions by MRI to ascertain a definite MS diagnosis. The CIS case with highest value was reported to have converted to definite MS. Primary progressive and relapsing remitting forms (PPMs and RRMs, respectively) showed dispersed values among which few revealed elevated, whereas SPMS patients showed rather grouped values. All MS groups showed positive and statistically strongly significant differences with the group of healthy individuals (p< 0.003 to p<0.0003).

Thus, the detection of pHERV-W ENV soluble Hexamer in sera from active MS cases of all clinical forms confirmed that it could possibly be released from demyelinating lesions into the bloodstream during active phases of the disease, despite unexplained conditions for this passage. At least a corresponding circulating antigen has now been discovered to be specifically detected in peripheral blood from MS patients, using the present methodology.

CONCLUSION

The present study has unexpectedly shown that pHERV-W Envelope antigen is expressed under different monomeric and oligomeric macro molecular structures.

Of particular interest, the hexameric form appears to be water-soluble and has unique properties allowing to specifically identify the form associated with actively demyelinating MS lesions.

Moreover, the unexpected results discovered from this novel analytic approach of pHERV-W ENV antigen study with capillary Immunoelectrophoresis and with specific conditions of protein extraction, along with separation by solubility and/or by size, made it possible to recover and detect the same soluble hexameric form in sera from MS.

Major learnings from the present study result from surprising discoveries that:

(i) the full length HERV-W envelope protein detected in MS demyelinating lesions has clearly different biochemical and antigenic properties from those of Syncytin-l, (ii) a unique water-soluble antigenic structure corresponding to a glycosylated envelope hexamer about 400KDa is dominantly detected in all MS lesions and, faintly, in one sample of MS NAWM but not in non-MS NAWM,

(iii) but, after deglycosylation, 100% specific detection of HERV-W soluble hexamer was observed in MS brain active lesions. (iv) the same hexameric soluble form was detected in MS sera with very high specificity, indicating its circulation into the bloodstream due to possible leakage from MS lesions under unexplained conditions to date.

Therefore, since a specific and clear-cut detection in sera was made possible with this newly developed protocol and yielded very significant differences compared to healthy controls in all forms or stages of MS disease, it provides a completely novel diagnostic technique for the detection of a new pHERV-W ENV antigen in blood, i.e. the soluble hydrophilic hexamer of PHERV-W ENV antigen.

In particular, this hexameric form is much more specifically detected that any other “global” antigenic source of pHERV-W antigen, as targeted with classical immunoassays or with alternative techniques assuming that a unique monomeric form would exist and falsely thinking that a monomer would be a soluble antigen. This is particularly relevant for its detection from peripheral blood, in particular in serum samples.

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Claims

1. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen formed by the HERV-W envelope protein in a biological fluid sample obtained from a patient suffering from an HERV-W associated disease

wherein the detection method comprises the following steps:

obtaining a soluble protein fraction from said biological sample; and

contacting and detecting the soluble oligomeric antigen with one or more ligand(s) targeting the SU region and/or the C terminal region of pHERV-W Envelope protein.

2. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to claim 1, wherein the step of contacting and detecting the soluble oligomeric antigen is performed by immunodetection and wherein the ligand targeting the pHERV-W Envelope SU region is an antibody targeting an epitope comprising or consisting in the amino acid sequence DLYNHY.

3. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to claim 1, wherein the step of contacting and detecting the soluble oligomeric antigen is performed by immunodetection and wherein the ligand targeting the C terminal of pHERV-W Envelope is an antibody targeting an epitope comprising or consisting in the amino acid sequence NDIEVTPP.

4. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of the preceding claims, wherein the step of obtaining a soluble protein fraction comprises:

extracting the total protein fraction, optionally in denaturing conditions; and obtaining a soluble protein fraction from said total protein fraction.

5. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of the preceding claims, wherein the method further comprises a step of size separation of proteins above 200, optionally 300 or optionally 350 kDa from the total protein fraction or from the soluble protein fraction.

6. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of the preceding claims, wherein the method further comprises a step of deglycosylation of the proteins, wherein said deglycosylation step can be performed on the total protein fraction or on the soluble protein fraction, or optionally on the proteins fraction above 100, 200, 300 or 350 kDa.

7. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of claims 2 and 4 to 6, wherein the method further comprises a step of contacting and detecting the soluble oligomeric antigen with a ligand targeting the C terminal region, optionally wherein the ligand targeting the C terminal of pHERV-W Envelope is an antibody targeting an epitope comprising or consisting in the amino acid sequence

NDIEVTPP.

8. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to claim 6 or 7, wherein the step of contacting and detecting the soluble oligomeric antigen with a ligand targeting the C terminal region is performed after the deglycosylation step.

9. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen in a biological fluid sample obtained from a patient, according to any one of the preceding claims comprising the following steps:

A:

extracting the total protein fraction from said biological sample,

obtaining a protein soluble hydrophilic fraction from the total protein fraction, and immunodetecting the soluble hydrophilic oligomeric antigen with an antibody as defined in claim 2;

optionally wherein the proteins of the total protein fraction or from the soluble protein fraction are further denatured, and optionally wherein the proteins from the total protein fraction or from the soluble protein fraction are further deglycosylated before the step of immunodetecting the soluble hydrophilic oligomeric antigen;

and/or

B. extracting the total protein fraction from said biological sample,

obtaining a protein soluble hydrophilic fraction from the total protein fraction immunodetecting the soluble hydrophilic oligomeric antigen with an antibody as defined in claim 3;

optionally wherein the proteins of the total protein fraction or from the soluble protein fraction are further denatured,

and wherein the proteins from the total protein fraction or from the soluble protein fraction are optionally further deglycosylated before the step of immune detecting the soluble hydrophilic oligomeric antigen.

10. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen in a biological fluid sample obtained from a patient, according to claim 9, wherein the sample is fractioned and wherein fractions of the biological fluid sample are submitted to the method of steps A and B.

11. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of claims 5-10, wherein the steps of size separation of the proteins and immunodetection are performed by capillary immunoelectrophoresis using one or more antibodies as defined in claim 2 or 3, optionally wherein the immunodetection is performed for proteins with an apparent molecular weight ranging from 200 to 500 kDa, optionally from

300 to 450 kDa.

12. An in vitro method for the detection of the soluble hydrophilic oligomeric antigen according to any one of the preceding claims, wherein the biological sample is patient’s serum, plasma, whole blood, blood cells, cerebro-spinal fluid, urine or saliva, optionally wherein the biological fluid sample is patient’s serum, plasma or whole blood.

13. An in vitro method for the diagnosis, the therapeutic monitoring, the prognosis of HERV- W associated diseases, or for stratifying patients suffering from an HERV-W associated disease, comprising performing the method according to any one of the preceding claims.

14. A method according to any of the preceding claims, wherein the HERV-W associated disease is selected from Multiple Sclerosis, diabetes, in particular type 1 diabetes, chronic inflammatory demyelinating polyradiculopathy (CIDP), Schizophrenia, bipolar disorder and cancer, preferably the HERV-W associated disease is multiple sclerosis.

15. A kit comprising:

- an antibody directed against the N-terminal portion of the HERV-W Env protein, notably targeting the SU region of the the N-terminal portion of the HERV-W Env protein, notably targeting an epitope comprising or consisting in the amino acid sequence DLYNHV; and

- an antibody targeting the C-terminal domain of the HERV-W Env protein, notably an antibody targeting an epitope comprising or consisting in the amino acid sequence

NDIEVTPP.