Title of Invention	PEPTIDE DOMAIN OF THE ENVELOPE OF A VIRUS WHICH INTERACTS WITH AN HASCT RECEPTOR
Abstract	The invention relates to a peptide domain required for interaction between the envelope of a virus pertaining to the HERV-W interference group and an hASCT receptor, comprising an N end point and a C end point. Said peptide domain is defined, at the N end point thereof, by a pattern formed by the amino acids L (Z)α-proIine-cysteine-X-cysteine in which Z is any amino acid, a is a whole number between 2 and 30, and X is any amino acid, and at the C end point thereof, by a pattern formed by the amino acids serine-aspartic acid-Xa-Xb-Xc-Xd-Xe-aspartic acid-Xf-Xg-(Z)β in which Xa-Xb-Xc-Xd-Xe-Xf-Xg are any amino acids, Z is any amino acid, β is a whole number between 15 and 25, preferably 20. The peptide domain comprises, between the N end point and the C end point, at least one patter selected from the following patterns; a pattern formed by the amino acids cysteine-tyrosine-X2-X3-X4-X5-X6-cysteine in which X2-X3-X4-X5-X6 are any amino acids, and a pattern formed by the amino acids cysteine- X7-X8-X9-X10-X11-X12-X13-X14-X15-cysteine-trytophane in which X7-X8-X9-X10-X11-X12-X13-X14-X15 are any amino acids.

Title of Invention

PEPTIDE DOMAIN OF THE ENVELOPE OF A VIRUS WHICH INTERACTS WITH AN HASCT RECEPTOR

Abstract

The invention relates to a peptide domain required for interaction between the envelope of a virus pertaining to the HERV-W interference group and an hASCT receptor, comprising an N end point and a C end point. Said peptide domain is defined, at the N end point thereof, by a pattern formed by the amino acids L (Z)α-proIine-cysteine-X-cysteine in which Z is any amino acid, a is a whole number between 2 and 30, and X is any amino acid, and at the C end point thereof, by a pattern formed by the amino acids serine-aspartic acid-Xa-Xb-Xc-Xd-Xe-aspartic acid-Xf-Xg-(Z)β in which Xa-Xb-Xc-Xd-Xe-Xf-Xg are any amino acids, Z is any amino acid, β is a whole number between 15 and 25, preferably 20. The peptide domain comprises, between the N end point and the C end point, at least one patter selected from the following patterns; a pattern formed by the amino acids cysteine-tyrosine-X2-X3-X4-X5-X6-cysteine in which X2-X3-X4-X5-X6 are any amino acids, and a pattern formed by the amino acids cysteine- X7-X8-X9-X10-X11-X12-X13-X14-X15-cysteine-trytophane in which X7-X8-X9-X10-X11-X12-X13-X14-X15 are any amino acids.

Full Text	Peptide domain required for interaction between the envelope of a virus pertaining to the HERV-W interference group and an hASCT receptor The present invention relates to a polypeptide domain responsible for interactions between a retroviral envelope of the HERV-W interference group and the receptors of the hASCT family. Human endogenous retroviruses (HERVs) constitute 8% of the human genome and are involved both in pathologies and in nonpathological phenomena. The human endogenous retrovirus family W (HERV-W) is derived from an infectious retroviral element that was integrated into the germ line 25 to 40 million years ago. the_HERV-W envelope protein, also called syncytin, is a fusogenic glycoprotein involved in the formation of the syncytiotrophoblastic layer of the placenta. It is encoded by the env gene of the proviral locus ERVWl and synthesized in the form of a gPr7 3 precursor which is specifically cleaved into two mature proteins, a surface subunit gp50 (SU) and a transmembrane subunit gp24 (TM). .In vitro, syncytin of the HERV-W- family induces a cell to cell fusion that is dependent on its interaction with a receptor-transporter of amino acids of the ASCT family (h-ASCT2, hASCTl). Phylogenic studies then showed that syncytin is related to a group of retroviruses comprising in particular the cat endogenous virus RD114, the monkey endogenous virus BaEV, simian retroviruses and avian retroviruses: avian reticuloendotheliosis virus REV-A and spleen necrosis virus SNV, all having in common the type 2 sodium-dependant neutral amino acid receptor-transporter or REPLACEMENT SHEET (RULE 26) ASC12 (rasko ex. al, 1999, Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2129-2134; Tailor et al, 1999 Journal of Virology, vol. 73(5) May 1999, P. 4470-4474). Thus, the infection of cells with viruses of this retrovirus group leads to a specific reduction in the transport of amino acids (Rasko et al., 1999). The infection of a cell with one of these retroviruses (or the expression of one of these envelopes in the cell) prevents, through interference (interaction) in relation to a receptor of the ASCT family, the infection of this same cell by another of these retroviruses or the fusion with another cell expressing another envelope. Through interference in relation to a receptor of the ASCT family, the infection of a cell by one of these retro¬viruses prevents the infection by another of these retroviruses. All these retroviruses belong to the same HERV-W virus interference group. The mechanisms of binding between the envelope and the ASCT receptor remain obscure, and to date no domain for binding to an ASCT receptor has been identified and defined either in the SU of the HERV-W envelope protein or in the SUs of retroviruses of the same interference group. This theme is nevertheless essential since the inhibition of the envelope/ASCT receptor interaction would in addition make it possible to prevent the entry of a retrovirus into the cell, and therefore to block its replication cycle, to block the phenomenon of envelope/ASCT receptor interaction and/or of cell fusion which may be involved in the formation of tumors, in the proliferation of metastasic cells or in drug resistance phenomena (see by way of illustration the publication "Cell fusion: A hidden enemy?. Cancer Cell: May 2003, vol. 3), to block the phenomenon of envelope/ASCT receptor interaction and/or of cell fusion which may be involved in nervous system diseases and even to inhibit the cell-cell fusion involved in trophoblastic differentiation (contraceptive vaccination). Furthermore, the inhibition of the envelope/hASCT receptor interaction could prevent tumor propagation by counteracting a local immunosuppression which may result from the envelope/hASCT receptor interaction. Indeed, it has been shown, on the one hand, that the infection of cells with viruses of this retrovirus group (in particular those inducing immunodeficiencies) leads to a specific reduction in the transport of amino acids (Rasko et al, PNAS, vol. 96. pp 2129-2134 (1999)), and on the other hand, a direct link is proposed between the impairment of the transport of amino acids and immunosuppression (Espinosa A, Villarreal LP., T-Ag inhibits implantation by EC cell derived embryoid bodies. Virus Genes. 2000; 20(3): 195-200; JE, Battini JL, Gottschalk RJ, Mazo I, Miller AD., The RD114/simian type D retrovirus receptor is a neutral amino acid transporter. Proc Natl Acad Sci USA, 1999, Mar 2; 96(5): 2129-34). Thus, as regards nervous system diseases, it is known that the hASCT receptors are involved in the specific transport of neutral amino acids and that neuronal cells, for the transmission of information, predominantly use neuromediators of a polypeptide nature. Thus, the binding of the Env-HERV-W protein to receptors which normally have to transport the amino acids required for the synthesis of neuromediators can affect the capacity of the neurons to synthesize the neuromediators by reducing the entry of the physiological agonists such as amino acids via the ASCT receptors. Moreover, if neurons whose intercellular networks form connections which are essential for the transmission of information circulating in the brain and the spinal cord, form syncytia following a fusion of several neurons which is induced by the Env-HERV-W protein, all the networks for transmission of information become disrupted and connected to the same fused "cellular package" and, furthermore, the neuromediator production activity of each cell is no longer individualized or connected to REPLACEMENT SHEET (RULE 26) the upstream or downstream conduction pathways (dendrites and axons) which are specific to it. Surprisingly, the inventors have identified the polypeptide region responsible for the interactions between the envelope of a virus, belonging to the HERV-W interference group and an hASCT receptor. To this effect, the present invention relates to a peptide domain necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, defined in that it starts with an N-terminus and ends with a C-terminus, and in that: ■ the N-terminus is defined by a motif, consisting of the amino acids (Z)a-proline- cysteine-X-cysteine in which Z is any amino acid a is an integer between 2 and 30 X is any amino acid, said motif being chosen from SEQ ID No. 1 to SEQ ID No. 29 and SEQ ID No. 44 to SEQ ID No. 72 the C-terminus is defined by a motif consisting of the amino acids serine-aspartic acid-Xg-Xb- Xc-Xd-Xe aspartic acid-Xf-Xg-(Z) p in which Xa, Xb, Xc, Xd, Xe, Xf, Xg are any amino acids Z is any amino acid P is an integer between 15 and 25, preferably 20 said motif being chosen from SEQ ID No. 30 to SEQ ID No. 40, and in that said peptide domain comprises, between the N-terminus and the C-terminus, at least one motif chosen from the following motifs: REPLACEMENT SHEET (RULE 26) a motif consisting of the amino acids cysteine- tyrosine-X2-X3-X4-X5-X6- cysteine, in which X2, X3, X4, X5, Xg are any amino acids said motif corresponding to SEQ ID No. 41 a motif consisting of the amino acids cysteine- X7-X8-X9-X10-X11-X12-X13-X14-X15-cysteine- tryptophan, in which X7, X8, X9, X10, X11, X12, X13, X14, X15 are any amino acids said motif corresponding to SEQ ID No. 42 or SEQ ID No. 73. The expression peptide domain according to the invention is understood to mean a minimum region of the envelope of a virus of the HERV-W interference group necessary for the recognition of an hASCT receptor. The peptide domains of the invention may be obtained by the genetic engineering technique which comprises the steps of: culturing a microorganism or eukaryotic cells transformed with the aid of a nucleotide sequence according to the invention, and recovering the peptide domain produced by said microorganism or said eukaryotic cells. This technique is well known to a person skilled in the art. For further details concerning it, reference may be made to the book below: Recombinant DNA Technology I, Editors Ales Prokop, Raskesh K Bajpai; Annals of the New York Academy of Sciences, Volume 646, 1991. The peptide domains of the invention may also be prepared by conventional peptide syntheses well known to a person skilled in the art. The expression interference group is understood to mean all the viruses for which the infection (expression) of a cell by one of its members prevents infection by ' REPLACEMENT SHEET (RULE 26) another member of the group by receptor interference. The expression any amino acid is understood to mean in particular an amino acid chosen from arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, asparagine, threonine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, cysteine, glycine, proline. The expression hASCT receptor is understood to mean any sodium-dependent neutral amino acid transporter. The expression motifs is understood to mean a succession of amino acids corresponding to a particular region of interest of the peptide domain according to the invention, which is expressed by all the viruses of the HERV-W virus interference group. Preferably, a is an integer between 3 and 18. Preferably X or Xaa of the Pro Cys Xaa Cys motif in SEQ ID No. 1 to SEQ ID No. 29 is an amino acid chosen from aspartic acid, glutamic acid, arginine: these sequences are those preferably chosen from SEQ ID No. 44 to SEQ ID No. 72. Preferably, Xa, Xb, Xc or the amino acids at positions 3, 4 and 5 of SEQ ID Nos. 30 to 40 are a glycine, Xa or the amino acid Xaa at position 6 of SEQ ID Nos. 30 to 4 0 is an amino acid chosen from proline and valine; Xe or the amino acid Xaa at position 7 of SEQ ID Nos. 30 to 40 is an amino acid chosen from glutamine, leucine and threonine; Xf or the amino acid Xaa at position 9 of SEQ ID Nos. 30 to 40 is an amino acid chosen from lysine, threonine, methionine and glutamine, Xg or the amino acid at position 10 of SEQ ID Nos. 30 to 40 is an amino acid chosen from alanine, lysine, isoleucine, REPLACEMENT SHEET (RULE 26) threonine and valine. Preferably, [3 is an integer equal to 20. Preferably, X2 or the amino acid Xaa at position 3 of SEQ ID No. 41 is an amino acid chosen from asparagine, threonine, glutamic acid, histidine, X3 or the amino acid Xaa at position 4 of SEQ ID No. 41 is an amino acid chosen from histidine, alanine, serine, lysine, glutamic acid; X4 or the amino acid Xaa at position 5 of SEQ ID No. 41 is an amino acid chosen from tyrosine, threonine, alanine, X5 or the amino acid Xaa at position 6 of SEQ ID No. 41 is an amino acid chosen from glutamine, arginine, threonine, Xe or the amino acid Xaa at position 7 of SEQ ID No. 41 is an amino acid chosen from leucine, glutamine, glutamic acid. Preferably X7 or the amino acid Xaa at position 2 of SEQ ID No. 42 is an amino acid chosen from proline, chreonine, arginine and asparagine; Xg or the amino acid Xaa at position 3 of SEQ ID No. 42 is an amino acid chosen from glycine, glutamic acid, asparagine, Xg 3r the amino acid Xaa at position 4 of SEQ ID No. 42 is an amino acid chosen from glycine, asparagine, soleucine, threonine, serine, Xio or the amino acid Xaa it position 5 of SEQ ID No. 42 is lysine or is deleted; k11 or the amino acid Xaa at position 6 of SEQ ID No. 42 ss an amino acid chosen from lysine, valine, soleucine, leucine, X12 or the amino acid Xaa at position 7 of SEQ ID No. 42 is an amino acid chosen ;rom glycine, asparagine; X13 or the amino acid Xaa at position 8 of SEQ ID No. 42 is an amino acid chosen from glutamine, lysine, valine, X14 or the amino acid kaa at position 9 of SEQ ID No. 42 is an amino acid thosen from valine, proline, serine, threonine, X15 or the amino acid Xaa at position 10 of SEQ ID No. 42 is in amino acid chosen from valine, isoleucine. REPLACEMENT SHEET (RULE 26) Preferably, the amino acid Xaa at position 2 of SEQ ID No. 73 is chosen from proline, threonine, arginine and asparagine, the amino acid Xaa at position 3 of SEQ ID No. 73 is chosen from glycine, glutamic acid, asparagine, the amino acid Xaa at position 4 of SEQ ID No. 73 is chosen from glycine, asparagine, isoleucine, threonine, serine, the amino acid Xaa at position 5 of SEQ ID No. 73 is chosen from lysine, valine, isoleucine, leucine, the amino acid Xaa at position 6 of SEQ ID No. 73 is chosen from glycine, asparagine, the amino acid Xaa at position 7 of SEQ ID No. 73 is chosen from glutamine, lysine, valine, the amino acid Xaa at position 8 of SEQ ID No. 73 is chosen from valine, proline, serine, threonine, the amino acid Xaa at position 9 of SEQ ID No. 73 is chosen from valine, isoleucine. Deletions are possible in the domains according to the invention indicated above. According to a particular embodiment of the invention, X10 is deleted. The invention also relates to a nucleotide sequence encoding a peptide domain according to the invention above. Such sequences may be prepared by chemical synthesis and genetic engineering using techniques well known to a person skilled in the art and described, for example, in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 198 9. The invention also relates to an epitope derived from the peptide domain according to the invention, characterized in that it induces an immune response against a virus belonging to the HERV-W interference REPLACEMENT SHEET (RULE 2 6) group. The expression epitope is understood to mean all or part of the peptide domain according to the invention recognized by a receptor located at the surface of a B or T lymphocyte or of a circulating antibody. The expression immune response is understood to mean all the biological mechanisms which allow . a pluricellular organism to maintain the coherence of the cells and tissues which constitute it and to ensure its integrity -in response to any attack which modifies the molecular structures of its constituents or which introduces foreign molecules into the organism. The invention also relates to a nucleotide sequence encoding an epitope as defined above. As indicated above, such sequences may be prepared by chemical synthesis and genetic engineering using techniques well known to a person skilled in the art and described, for example, in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 1989. The invention also relates to an expression vector characterized in that it comprises a nucleotide sequence according to the invention, and the means necessary for its expression. By way of expression vector, there may be mentioned, for example, plasmids, viral vectors of the vaccinia virus, adenovirus, baculovirus, poxvirus or retrovirus type, bacterial vectors of the salmonella or BCG type. The expression means necessary for its expression is understood to mean any means which make it possible to obtain a peptide from a nucleotide sequence, such as in particular a promoter, a transcription terminator, a replication origin and preferably a selectable marker. REPLACEMENT SHEET (RULE 26) The vectors of the invention may also comprise sequences necessary for targeting peptides to particular cell compartments. The invention also relates to a host microorganism or cell transformed with at least one expression vector according to the invention. By way of examples of microorganisms which are suitable for the purposes of the invention, there may be mentioned yeasts, such as those of the following families: Saccharomyces, Schizosaccharoyces, Kluveromyces, Pichia, Hanseluna, Yarowia, Schwaniomyces, Zygosaccharomyces; Saccharomyces cerevisiae, Saccharomyces carlshergensis and Kluveromyces lactis being preferred; and bacteria such as E.' coli and those of the following families: Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus _a.ndi Streptomyces. By way of examples of transformed host cells, there may be mentioned cells derived from animals such as mammals, reptiles, insects and the like. The preferred eukaryotic cells are cells derived from the Chinese hamster (CHO cells), from monkeys (COS and Vero cells), from young hamster kidney (BHK cells), from pig kidney (PK 15 cells) and from rabbit kidney (RK13 cells), human osteosarcoma cell lines (143 B cells), human HeLa cell lines and human hepatoma cell lines (of the Hep G2 cell type), and insect cell lines (for example Spodoptera frugiperda), a human embryonic kidney cell line (for example HEK293T). The host cells may be provided in cultures in suspension or in flasks, in tissue cultures, organ cultures and the like. The invention also relates to an antibody directed against a peptide domain according to the invention or REPLACEMENT SHEET (RULE 2 6) against an epitope according to the invention. The expression antibody is understood to mean both a whole antibody and an antibody fragment. The recombinant antibodies may be obtained according to conventional methods known to a person skilled in the art, from prokaryotic organisms, such as bacteria, or from eukaryotic organisms, such as yeasts, mammalian, plant, insect or animal cells, or by extracellular production systems. The monoclonal antibodies may be prepared according to conventional techniques known to a person skilled in the art such as the hybridoma technique whose general principle is recalled below. In a first instance, an animal, generally a mouse (or cultured cells in the context of in vitro immunizations) , is immunized with a target antigen of interest, whose B lymphocytes are then capable of producing antibodies against said antigen. These antibody-producing lymphocytes are then fused with "immortal" myelomatous cells (murine in the example) in order to give rise to hybridomas. From the heterogenous mixture of cells thus obtained, a selection of the cells capable of producing a particular antibody and of multiplying it indefinitely is then carried out. Each hybridoma is multiplied in clone form, each leading to the production of a monoclonal antibody whose recognition properties in relation to the antigen of interest may be tested, for example by ELISA, by one-or two-dimensional immunotransfer, by immuno¬fluorescence or with the aid of a biosensor. The monoclonal antibodies thus selected are subsequently purified in particular according to the affinity chromatography technique. REPLACEMENT SHEET (RULE 26) The expression antibody fragment is understood to mean any antibody fragment following an immune response against a virus belonging to the HERV-W interference group. These antibody fragments may, for example, be obtained by proteolysis. Thus, they may be obtained by enzymatic digestion, resulting in fragments of the Fab type (treatment with papain; Porter RR, 1959, Biochem. J., 73: 199-125) or of the F(ab)'2 type (treatment with pepsin; Nisonoff A. et al., 1960, Science, 132: 1770-1771) . They may also be prepared by the recombinant route (Skerra A., 1993, Curr. Opin. Immunol., 5: 256-262). Another antibody fragment which is suitable for the proposals of the invention comprises an Fv fragment which is a dimer consisting of the noncovalent combination of the variable light (VL) domain and of the variable heavy (VH) domain of the Fab fragment, and therefore the combination of two polypeptide chains. In order to improve the stability of the Fv fragment due to the dissociation of the two polypeptide chains, this Fv fragment may be modified by genetic engineering by inserting a suitable linker peptide between the VL domain and the VH domain (Huston P. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5879-5883). The expression scFv fragment ("single chain Fragment variable") is then used because it consists of a single polypeptide chain. The use of a linker peptide preferably composed of 15 to 25 amino acids makes it possible to link the C-terminus of one domain to the N-terminus of the other domain, thus constituting a monomeric molecule endowed with binding properties similar to those of the antibody in its complete form. Both orientations of the VL and VH domains are suitable (VL-linker-VH and VH-linker-VL) because they exhibit identical functional properties. Of course, any fragment known to a person skilled in the art and exhibiting the immunological characteristics defined above are suitable for the purposes of the invention. REPLACEMENT SHEET (RULE 2 6) The invention also relates to the use of at least one peptide domain according to the invention, of at least one epitope according to the invention, of at least one antibody according to the invention or of at least one nucleotide sequence according to the invention, for the preparation of a medicament intended for the inhibition, prevention or treatment of an infection caused by a virus belonging to the HERV-W interference group in an animal, preferably humans. The peptide domain according to the invention may be used in particular for targeting cells expressing a receptor of the hASCT family in order to transduce a signal, and modulate the flow of amino acids (cancer treatments) . The expression elements necessary for a constitutive expression of peptides is understood to mean a ubiquitous promoter specific to eukaryotic cells. By way of elements necessary for an inducible expression of the peptides, there may be mentioned the elements for regulating the E. coli operon for resistance to tetracycline (Gossen M. et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992)). The use of at least one peptide domain according to the invention, of at least one epitope according to the invention, or of at least one nucleotide sequence according to the invention is particularly suitable for the preparation of a medicament intended for the prevention of an infection caused by a virus belonging I to the HERV-W interference group in an animal, preferably humans. The use of at least one antibody according to the invention is particularly suitable for the preparation of a medicament intended for the inhibition or treatment of an infection or a pathology induced by a virus belonging to the HERV-W interference group in an animal, preferably humans. The invention also relates to a pharmaceutical REPLACEMENT SHEET (RULE 26) composition comprising, by way of active substance, at least one peptide domain according to the invention, at least one epitope according to the invention, or alternatively at least one of the nucleotide sequences according to the invention, in particular placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, in combination with a pharmaceutically appropriate vehicle. The invention also relates to a pharmaceutical composition comprising, by way of active substance, at least one antibody according to the invention, in combination with a pharmaceutically appropriate vehicle. Of course, persons skilled in the art will easily determine the pharmaceutically appropriate vehicle and the quantity of peptide domains, of epitopes, of nucleotide acids or of antibodies to be used according to the constituents of the pharmaceutical composition. In the pharmaceutical compositions according to the invention, for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, rectal or transdermal administration, the active substance may be administered in unit forms for administration or as a mixture with conventional pharmaceutical supports and intended for administration by the oral route, for example in the form of a tablet, a gelatin capsule, an oral solution, and the like, or by the rectal route, in the form of a suppository, or by the parentral route, in particular in the form of a solution for injection, in particular by the intravenous, intradermal or subcutaneous route, and the like, according to conventional protocols well known to persons skilled in the art. For topical application, the active substance may be used in creams, ointments, lotions, eyedrops. REPLACEMENT SHEET (RULE 26) When a solid composition in tablet form is prepared, the active substance is mixed with the pharmaceutically acceptable excipient, also called a pharmaceutical vehicle, such as gelatin, starch, lactose, magnesium stearate, talc, gum Arabic or the like. The tablets may be coated with sucrose, with a cellulose derivative or with other appropriate materials. It is also possible to treat them such that , they have a prolonged or delayed activity and they continuously release a predetermined quantity of the active substance. It is also possible to obtain a preparation as gelatin capsules by mixing the active substance with a diluent and pouring the mixture into soft or hard gelatin capsules. It is also possible to obtain a preparation in syrup form or for administration in the form of drops, in which the active substance is present together with a sweetener, an antiseptic, such as in particular methylparaben and propylparaben, and a taste enhancer or an appropriate colorant. The powders or water-dispersible granules may contain the active substance in the form of a mixture with dispersing agents or wetting agents, or suspending agents, well known to persons skilled in the art. For parentral administration, aqueous suspensions, isotonic saline solutions or sterile solutions or solutions for injection which contain dispersing agents, pharmacologically compatible wetting agents, such as in particular polyethylene glycol or butylene glycol, are used. The medicament or the pharmaceutical composition according to the invention may additionally comprise an activating agent which induces the effects of a medication or reinforces or supplements the effects of the principal medication, by increasing in particular the bioavailability of the principal medication. The dosage depends on the seriousness of the condition REPLACEMENT SHEET (RULE 26) and will be adapted according to a conventional protocol. As a guide, when the active substance is a monoclonal antibody, the weekly dose is from 1 to 10 mg/kg, in combination with a pharmaceutically acceptable excipient. The invention also relates to a diagnostic composition for the detection and/or quantification of a virus belonging to the HERV-W interference group, or the detection and/or quantification of an immune response against said virus, comprising at least one peptide domain according to the invention, at least one epitope according to the invention, at least one of the nucleotide sequences according to the invention, or at least one antibody according to the invention. A diagnostic composition comprising at least one peptide domain according to the invention, at least one epitope according to the invention, at least one of the nucleotide sequences according to the invention, is particularly suitable if it is desired to determine if as apatient has an immune response against a virus belonging to the HERV-W interference group while a diagnostic composition comprising at least one antibody according to the invention is particularly suitable for the detection and/or quantification of a virus belonging to the HERV-W interference group. The invention also relates to a method for the detection and/or quantification of a virus belonging to the HERV-W interference group in a biological sample taken from an individual liable to be infected by said virus, characterized in that it comprises the steps consisting in: bringing said biological sample into contact with at least one antibody according to the invention under conditions allowing the formation of a complex between the virus and REPLACEMENT SHEET (RULE 26) the antibody, and detecting and/or quantifying the formation of said complex by any appropriate means. The expression biological sample is understood to mean a biological sample of human or animal origin liable to contain said virus, such as a sample of blood, plasma, serum, urine, cerebrospinal fluid, or of tissues, such as placenta, testicles, prostate and breast. The step of bringing into contact is a step that is conventionally known to a person skilled in the art. The detection/quantification step may be carried out by any detection means known in the field of immunological assays of very small molecules, such as direct detection, that is to say without the intermediary of a binding partner or of binding partners, and indirect detection, that is to say through the intermediary of a binding partner or of binding partners. The direct detection of the binding between the antibody or antibody fragment of the invention and the virus may be carried out for example by surface plasmon resonance or by cyclic voltammetry on an electrode bearing a conducting polymer. In this case, the antibody of the invention serves to immunocapture all or part of the virus, which is then eluted. The elution may be carried 'out by any elution method known to a person skilled in the art, such as a pH shock. In the case of indirect detection, the second step of the method of the invention may be carried out according to the conventional ELISA competition assay technique. The antibody of the invention then serves as binding partner serving to capture all or part of the virus in the sample. The detection may then be performed by competition between all or part of the REPLACEMENT SHEET (RULE 2 6) virus which may be contained in the sample to be tested and a previously labeled known quantity of virus. The expression labeling is understood to mean the attachment of a marker capable of directly or indirectly generating a detectable signal. A nonlimiting list of these markers consists of: ■ enzymes which produce a detectable signal, for example, by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, acetylcholine esterase, P- galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as luminescent, coloring compounds, radioactive molecules such as 32P, 35S or 1251, fluorescent molecules such as fluorescein, rhodomine, alexa or phycocyanins, and particles such as gold or magnetic latex particles, liposomes. ndirect labeling systems may also be used, such as, for example, via another ligand/anti-ligand pair. The igand/anti-ligand pairs are well known to a person killed in the art, and the following pairs may be entioned for example: biotin/streptavidin, biotin/avidin, apten/antibody, antigen/antibody, peptide/antibody, agar/lectin, polynucleotide/complementary strand for the plynucleotide. In this case, it is the ligand which is ound to the binding partner. The anti-ligand may be etected directly by the markers described in the roceeding paragraph or may itself be detected by a igand/anti-ligand. hese indirect systems may lead, under certain anditions, to an amplification of the signal. This ignal amplification technique is well known to a erson skilled in the art, and reference may be made to he previous patent applications FR98/10084 or REPLACEMENT SHEET (RULE 2 6) WO95/08000 by the applicant or to the article J. Histochem. Cytochem., (1997), 45: 481-491. The labeling of molecules is widely known to a person skilled in the art and is described for example by Greg T. Hermanson in Bioconjugate Techniques, 1996, Academic Press Inc., 525B Street, San Diego, CA92101 USA. Depending on the type of labeling used, such as for example using an enzyme, a person skilled in the art will add reagents which allow visualization of the labeling. Such reagents are widely known to a person skilled in the art and are described in particular .in Principles and Practice of Immunoessay, 2nd edition, Edited by C. Price, D.G. Newman, Stockton Press, 1997, 345 Park Avenue South, New York. The invention also relates to the use of the above composition for the in vitro screening of a virus belonging to the HERV-W interference group in a biological sample or specimen. In particular, the early screening of a virus such as SRVl and SRV2, viruses that are involved in immunodeficiency mechanisms in monkeys, makes it possible to provide a treatment suitable for the host before the appearance of an immunodeficiency. Moreover, the early screening of HERV-W, involved in placental pathologies, makes it possible to modulate its expression, for example, during a preeclampsia. The invention also relates to the use of a peptide domain according to the invention or of an epitope according to the invention, or of an antibody according to the invention, for inhibiting the interaction between the envelope of a virus belonging to the HERV-W REPLACEMENT SHEET (RULE 2 6) interference group and an ASCT receptor. This makes it possible, in particular, to obtain a contraceptive immunotherapy. The invention also relates to the use of a peptide domain according to the invention for identifying chemical or biological molecules whose interaction with all or part of this peptide domain blocks the interaction between the envelope of a virus belonging to the HERV-W interference group and an ASCT receptor. For example, when a peptide domain according to the invention of HERV-W is used, this makes it possible to obtain in particular chemical or biological molecules that are highly suitable in order to obtain a contraceptive treatment. The use of such chemical mole¬cules to inhibit the interaction between the envelope of a virus belonging to the HERV-W interference group and an ASCT receptor is of therapeutic interest. As a guide, two generic methods allowing the screening of chemical or biological molecules capable of inhibiting the env/receptor interaction are described below. In a context where it is possible to produce a soluble envelope, it is advisable to determine if a chemical or biological molecule alters the env/receptor interaction according to an ELISA type method using cells expressing at least one hASCT receptor in a capture phase. Thus, on a 96-well plate, cells expressing an hASCT receptor of interest are cultured or adsorbed, and an env/receptor interaction is detected via the use of a labeled soluble envelope (histine tag, GPF fusion), and therefore capable of generating a reference signal that can be assayed. If a signal reduction is observed after preincubation of said soluble envelope with a chemical or biological molecule, that means that the chemical or biological REPLACEMENT SHEET (RULE 26) molecule alters the env/receptor interaction. Alter¬natively, it is possible to use a retroviral vector pseudotyped by the envelope of interest and expressing a detectable marker (LacZ) and to carry out the same test. It is also possible to select molecules of interest via a measurement of fusion inhibition. Cells expressing the receptor of interest (cell-recept), for example HeLa or XC-RDR cells, and cells constitutively expressing a marker (for example, LacZ) and transiently or stably expressing the envelope of interest (cell-env-LacZ) are used; the envelope of interest was modified beforehand at the level of its intra-cytoplasmic tail by exchange with the intracytoplasmic domain of HERV-W env so as to make it constitutively fusogenic (Cheynet et al, 79(9): 5586-5593, 2005). The bringing into contact of the two cell types, "cell-recept" in excess and "cell-env-LacZ" in insufficient amount, leads to the formation of multinucleated giant cells or syncytia, containing one or two blue nuclei derived from "cell-env-LacZ" and tens of white nuclei derived from "cell-recept". An identical co-culture performed in the presence of a chemical or biological molecule altering the env/receptor interaction leads to a reduction in the number of syncytia and their nucleus content. Automation of such measurements with the aid of a CCD camera is possible. The invention also relates to the use of a peptide domain in accordance with the invention for generating antibodies blocking . the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor. The invention also relates to a method for determining a polypeptide region necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, characterized in that: REPLACEMENT SHEET (RULE 26) the nucleotide and/or peptide sequence of the precursor envelope of said virus is identified the signal part is excluded a serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic acid-Xf-Xg is detected domain in which Xa, Xb, Xc, Xd, Xg, Xf, Xg, are any amino acids which correspond to SEQ ID No. 43 the C-terminus is excluded between 15 and 25 amino acids, preferably 20 amino acids, after said serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic acid-Xf-Xg domain, which corresponds to SEQ ID No. 43. Preferably, Xa, Xb, Xc is an amino acid which is glycine, Xd is an amino acid chosen from proline and valine, Xg is an amino acid chosen from glutamine, leucine and threonine, Xf is an amino acid chosen from lysine, threonine, methionine and glutamine, Xg is an amino acid chosen from alanine, lysine, isoleucine, threonine and valine. For the purposes of the present invention, the nucleotide sequence and/or peptide consequent of the precursor envelope of said virus is identified by any means known to a person skilled in the art, who may refer in particular to Maniatis (ed. 1989). The signal part is excluded by any means known to a person skilled in the art, as described in particular in "Improved Prediction of Signal Peptide: SignalP 3.0" Jannick Dyrl0v Bendtsen, Henrik Nielsen, Gunnar von Hiejne and S0ren Brunak, J. Mol. Biol., 340: 783-795, 2004. Said domain is detected by any means known to a person skilled in the art, that is to say using Blast or Fasta type software (see, in particular, Altschul SF, Gish W, REPLACEMENT SHEET (RULE 26) Miller W, Myers EW, Lipman DJ., Basic local alignment search tool. J. Mol. Biol. 1990 Oct 5; 215(3): 403-10). The invention also relates to a peptide domain capable of being obtained by the above method. The accompanying figures are given by way of explanatory example and have no limiting character. They will make it possible to better understand the invention. figure 1 illustrates the phenotypical characteristics and properties of soluble recombinant proteins derived from Env-W. In particular, figure la illustrates the Largest soluble recombinant protein comprising all or part of the SU and TM subunits (Env-Gp60) and the soluble recombinant protein corresponding to the SU subunit (EnvSU). Figure lb represents the flow cytometry analysis of the test for binding of the EnvSU recombinant protein to XC hASCT2 and XC hASCTl cells expressing the hASCT2 and hASCTl receptors, respectively. Figure 1c illustrates the test of interference of binding to the TE671 cells (control ASCT2), TE671RD cells (blocked hASCT2) and TE671galv cells (blocked Pitl) . Figure 2 illustrates the definition of the minimum oinding domain of the ERV-W envelope to the hASCT2 receptor (RBD for receptor binding domain). In particular, figure 2a describes all the deletion mutants designed from EnvSU. Figure 2b represents the flow cytometry analysis of the test of binding of the recombinant proteins derived from EnvSU to the XC hASCT2 cells expressing the hASCT2 receptor, in particular the binding of the Envl97, Envl68 and Envl44 Tiutants and the binding defect of the Env69-317, Envl69-317 and Envll7 mutants. REPLACEMENT SHEET (RULE 26) Figure 3a illustrates the definition of an immunogenic peptide inside the domain according to the invention (RBD) corresponding to region 21-144 of the precursor of the HERV-W envelope protein. Figure 3b shows the inhibition of the binding of the RBD to its receptor with the aid of an antibody produced from the immunogenic peptide (antiSU-EnvW) and the absence of inhibition of RBD-receptor binding in the presence of a nonspecific antibody (antlTM-EnvW). Figure 4 represents the alignment of the retroviral envelope sequences belonging to the same interference group and shows the boundaries of the signal peptide, of the SU (surface unit) subunit and of the TM (Trans membrane) subunit and the receptor binding site. The sequences are HERV-W (Human Endogenous Retroviral Family W), RD114 (Cat Endogenous retrovirus), REV (Avian Reticuloendotheliosls Virus), BAEV (Baboon endogenous virus (strain M7)), SRVl (Simian retrovirus SRV-1), SRV2 (Simian retrovirus SRV-2) and MPMV (Simian Mason-Pfizer virus). The following examples are given by way of illustration and have no limiting character. They will make it possible to better understand the invention. Example 1: Molecular and phenotypical characterization of recombinant envelopes Construction and production of the HERV-W envelope SU subunit A vector phCMVEnv-Gp60 allowing the expression of a soluble recombinant envelope protein was designed from the expression vector phCMV-Env-W (Blond J Virol, Vol 74(7): 3321-3329, 2000) containing the HERV-W envelope gene (538 amino acids) (clone PH74, Blond et al. J Virol Vol 73(2): 1175-1185, 1999). REPLACEMENT SHEET (RULE 26) The soluble envelope (Gp60,1-435) was constructed as described below: (1) The native cleavage site RNKR (AA 314 to 317) between the SU and TM subunits was mutated to AAAR in order to allow the production of a fusion protein that was stable and not of two SU-TM subunits cleaved and then recombined by a disulfide bridge. (2) The transmembrane (tm) and intracytoplasmic (CYT) regions corresponding to amino acids 436 to 538 were deleted in order to obtain a soluble protein. (3) A spacer arm having the composition (GGGS)3, followed by a polyhistidine tail (RGS-HHHHHH), were added at the C-terminal position in order to allow the purification of this protein by IMAC and the detection by an anti-histidine monoclonal antibody (Qiagen, RGS H6) . Starting with the vector phCMVEnv-Gp60 expressing the soluble envelope, the vector phCMV-EnvSU was constructed, allowing the production of an SU protein. The soluble SU is a fusion protein containing a C-terminal polyhistidine tail having the sequence RGS-HHHHHH immediately downstream of the sequence AAAR, in order to allow the purification of this protein by IMAC and the detection by an anti-histidine monoclonal antibody (Qiagen, RGS H6) . The schematic structure of the various proteins produced from the vectors phCMV-Env-W, phCMV-EnvGp60 and phCMV-EnvSU is illustrated in figure la. Production of the soluble envelope The expression plasmid phCMV-EnvGp60 or phCMV-EnvSU is transfected into the HEK293T cells by precipitation with calcium phosphate. The supernatant containing the GP60 or SU envelope is collected after 48 hours of REPLACE^ "" production in a serum-free medium and filtered on 0.45 µm membranes in order to remove the cellular debris. 20 µ1 of supernatant are directly analyzed on a polyacrylamide gel and by Western blotting with an anti-histidine monoclonal antibody (Quiagen, RGS H6) . The GP60 and SU proteins are correctly expressed in soluble form. Binding test and analysis by flow cytometry The stable lines XChASCT2 and XChASCTl constitutively expressing the hASCT2 (XChASCT2) or hASCTl (XChASCTl) receptors were established after transfection of XC cells (rat sarcoma) with vectors expressing either human receptor hASCT followed by selection of a clone as described above (Frendo et al. , Mol. Cell Biol,, Vol 23(10): 3566-3574, 2003). The following human cells are described in Blond J Virol, Vol 74(7): 3321-3329, 2000. The TE671 cells express hASCT2. The TE671RD cells constitutively express the RD114 envelope (cat endogenous retrovirus) belonging to the same interference group and therefore recognizing the hASCT2 receptor. TE671galv cells constitutively express the GALV (gibbon ape leukemia virus) envelope belonging to another interference group and recognizing the PiT1 receptor. The cells were washed in PBS and harvested by detaching with 0.02% versene in PBS. A total of 10 cells were incubated with 1 ml of filtered supernatant containing the soluble envelope (Gp60 or SU) for 1 hour at 37°C. The cells were washed with PBA (PBS and 0.5% sodium azide) containing 2% fetal calf serum and were labeled for 1 hour at 4°C with an anti-histidine monoclonal antibody (RGSH6, Quiagen). The cells were washed once with PBA and incubated with a secondary antibody coupled to fluorescein isocyanate for 1 hour at 4°C. The cells were washed twice with PBA and analyzed by REPLACEMENT SHEET (RULE 2 6) flow cytometry. Using the target cells XChASCT2, the inventors demonstrated that the recombinant protein Gp60 corresponding to a soluble form of the envelope has a phenotypical characteristic identical to that of the wild-type envelope, namely that it is capable of binding to XC cells expressing the hASCT2 receptor. Using the target cells XChASCT2, XChASCTl, TE671, TE671RD, TE671 galv, the inventors demonstrated that the recombinant protein corresponding to the SU subunit of the envelope exhibits phenotypical characteristics identical to those of the wild-type envelope. First of all, the SU subunit is capable of binding to two receptors hASCTl and hASCT2 (figure lb). Furthermore, this protein was tested in relation to human cells TE671 and derived cells TE671RD and TE671galv. The soluble SU protein bound to the TE6781 cells expressing the hASCT2 receptor and the TE671galv cells blocked for the PiTl receptor, but did not bind to the TE671RD cells blocked for the hASCT2 receptor (figure Ic) . The SU recombinant protein and the envelope of the RD114 retrovirus specifically interfered. Example 2: Identification of the domains for interaction of the SU part of the W envelope with its hASCT2 receptor In order to identify the boundaries of the region of the envelope binding to the hASCT2 receptor, the inventors constructed a set of deletion mutants from the N- and C-terminal ends. The domains of the SU subunit were obtained by PCR and subcloned into the expression vector pHCMV-EnvSU and sequenced. The expression plasmids phCMV-EnvSU, Env69-317, Envl97, Envl68, Envl69-317, Envll7 and Envl44 (figure 2a) were transfected into the HEK293T cells by precipitation REPLACEMENT SHEET (RULE 26) with calcium phosphate. The conditions for production and analysis of the proteins are identical to those detailed in example 1. EnvSU, Env69-317 and Envl97 proteins were correctly expressed in soluble form. Using the XC cells constitutively expressing hASCT2, the inventors emonstrated that the Envl97 protein was capable of ending to the receptor expressed at the surface of the clls like the SU subunit (1-317). Thus, the first 176 esidues of the mature SU subunit (and therefore acking its signal peptide) were sufficient for binding a the surface of the cells expressing the hASCT2 eceptor. The deletion of the 21-68 region resulted in loss of binding to the receptor also indicating its evolvement in the receptor binding domain (RBD) . On he other hand, the truncated Envl68 protein showed a power capacity for binding to the hASCT2 receptor. order to obtain the equivalent quantities in the upernatants between the various truncated proteins, the inventors fused two smaller domains of the -terminal region of SU (Envll7 and Envl44) to the -terminal region of the SU subunit (Envl69-317), the atter domain not binding to hASCT2. The level of kpression of the Envll7 and Envl44 proteins was Lmilar and the proteins were expressed in soluble form. The binding test showed that only the Envl44 rotein was capable of binding to the cells expressing he hASCT2 receptor. The absence of binding of the nvll7 protein to the surface of the cells indicated he loss of at least one determinant of binding inside the 117-144 region. pnsequently, the boundaries of the domains for proteraction of the W envelope with its receptor are efined by amino acids 21 to 144. REPLACEMENT SHEET (RULE 26) It should be noted that, in general, proteins (including the envelope proteins) intended for secretion or for membrane expression are synthesized in the granular endoplasmic reticulum (ER) . The translocation of the neosynthesized proteins in the ER is conditioned by an N-terminal signal peptide (Walter and Lingappa 1986). The hydrophobic region of the signal peptide initiates penetration into the membrane of the reticulum, bringing behind it the remainder of the neosynthesized peptide. Since the translocation starts at the same time as the synthesis, it is the peptide being translated which crosses the ER membrane. While the protein passes into the lumene of the ER, the signal sequence is cleaved by a specific cellular enzyme, signal peptidase (Walter P, Johnson AE: Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 1994, 10: 87-119). The translocation of Env into the ER is stopped by the (hydrophobic) transmembrane domain of the glycoprotein which becomes anchored at the phospholipid membranes. In the ER lumene, the regions (SU and part of TM) intended to become extracytoplasmic are folded (the disulfide bridges formed), glycosylated and oligomerized. After oligomerization, the proteins undergoing maturation are transported into the Golgi apparatus where they undergo new glycan maturation processes and cleavage by furin-type endoproteases recognizing a motif R/KXXR leading to two SU and TM subunits. The mature protein is targeted to the plasma membrane by virtue of a motif present on the intracytoplasmic tail containing a tyrosine (aliphatic/aromatic(Y-X-X)). Example 3: Test of In vitro inhibition of the binding of the envelope to its receptor (definition of a peptide and generation of a rabbit antibody) REPLACEMENT SHEET (RULE 2 6) A peptide (112-129, TGMSDGGGVQDQAREKHV+C, 19 amino acids) was defined from the region defined in example 2 and from a determination of the potentially antigenic regions of the SU subunit. A cysteine was added at the C-terminal position for the KLH (keyhole limpet hemocyanin, cf. Frendo et al., Mol. Cell Biol. Vol 23(10): 3566-3574, 2003) coupling. This peptide was used to immunize a rabbit and then to affinity purify the polyclonal antibody directed against the region 112-119 contained in the serum of this rabbit. The Envl44 protein is preincubated at 37°C for one hour with either the anti-SU polyclonal antibody or with an anti-TM polyclonal antibody. The formation of the Envl44 protein-anti-SU antibody complex drastically reduced the binding of the envelope to the cells expressing the hASCT2 receptor. By contrast, the use of an antibody not directed against the RBD did not adversely affect its binding to the hASCT2 receptor. Example 4: Production of monoclonal antibodies directed against the HERV-W envelope protein Immunization of mice with DNA Three female six-week-old BALB/c mice (IFFA-Credo) were immunized by direct injection of naked plasmid DNA (phCMV-env-W) containing the gene for the HERV-W envelope. The injections were performed by the intradermal route with the aid of a gene gun. Five injections of 2 µg of DNA were first performed for each mouse followed by a booster with two injections of 4 µg of DNA. The sera were collected and the antibody titer for each serum was determined. Since the antibody titer was too low, a cellular lysate was prepared. Preparation of the cellular lysate REPLACEMENT SHEET (RULE 26) The rhabdomyosarcoma cells TelCeB6 (ATCC CRL8805) were transfected with the plasmid phCMV-env-W. After about 20 hours and the presence of syncytia, a cellular extract was prepared in PBS buffer containing 0.5% Triton. The protein extracts were assayed by Bradford. The env-W antigen concentration corresponded to 9.5 pg/yl of total proteins. Immunization of mice with an extract of cellular lysate The same mice first of all received an injection of 10 ]jg of cellular lysate by the intraperitoneal route followed by a booster injection of 2 x 100 µg of cellular lysate by the intraperitoneal route. Three days before the fusion with myeloma cells, another injection was performed,-by the intravenous route, with 22 µg of soluble envelope protein Gp60 obtained from the plasmid phCMV-Env-Gp60 as described in example 1, purified beforehand, before injection, on to an Ni-NTA resin (Quiagen) according to the following conditions: binding in phosphate buffer pH 8, washes in phosphate buffer pH 8 and in ammonium acetate pH 6, elution in ammonium acetate buffer pH 3.5 and concentration with speed vac. 47 jjg of the eukaryotic Gp60 protein thus obtained were reserved for the injection by the intravenous route described above. After fusion, the hybridoma supernatants were tested by immuno¬fluorescence on the transfected and bound cells (TeLCeb6), the antibodies were screened by a functional ELISA test using the Env-W protein at a concentration of 9.2 µg/µl of total proteins and an Env AS protein as negative control at a concentration of 13.3 µg/µl of total proteins and the most effective antibodies were selected. The monoclonal antibodies 2H1H8, '12C7A3 and IF11B10 were thus obtained. The monoclonal antibodies 2H1H8 and 12C7A3 are directed against the nonglycosylated N- REPLACEMENT SHEET (RULE 26) terminal part of the SU region of the Env-HERV-W proteins. They are directed against the RDB as shown by a Western blot assay with the aid of Env 144. The monoclonal antibody IF11B10 is directed against the glycosylated C-terminal part of the SU region of the Env-HERV-W protein as shown by a Western blot assay with the aid of Env 169-317. It does not recognize Env 144. Example 5: Test of in vitro inhibition of the binding of the envelope to its receptor and inhibition of the formation of syncytia with the aid of monoclonal antibodies by a cell to cell fusion test (coculture) The plasmid for expressing the envelope glycoprotein is transfected into the cells TELCeB6 by precipitation with calcium phosphate at two quantities 100 and 500 ng (Cosset-et al., Journal of Virology, 69 (10): 6314-6322 (1995)). The cells expressing the envelope are detached from the support 20 hours after transfection and are preincubated at 37 °C for one hour, respectively, with the anti-HIV 23A5 monoclonal antibody, the anti-TM Env-HERV-W 6A2B2 monoclonal antibody (previously obtained), the anti-SU Env-HERV-W 2H1H8 12C7A3 and IF11B10 monoclonal antibodies (dilution l/50th). Next, they are reinoculated at equal concentration (0.4 x 105 cells/well) into 12-well plates. Epithelioid-carcinoma-indicating human cells (Hela, ATCC CCL-2) are then added to the transfected cells in an amount of 2 X 10^ cells per well and the co-culture is continued for 24 h. An XGal (5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside) staining may then be performed in order to stain the nucleus of the cells TELCeB6 (Cosset et al., Journal of Virology, 69 (10): 6314-6322 (1995) ) . It is followed by staining with May-Griinwald and Giemsa solutions (MERCK) performed according to the suppliers' recommendations. The fusion observed corresponds to a fusion "from within", that is to say a REPLACEMENT SHEET (RULE 26) cell to cell fusion, starting with a cell expressing the envelope, in contrast to a fusion "from without" which corresponds to a formation of syncytia following a virion-cell (s) fusion. The results presented in table 1 below express the number of fused cells counted. The results presented above show that the formation of the Env protein-anti-SU 2H1H8 and 12C7A3 antibody complex dramatically reduces the binding of the envelope to cells expressing the hASCT2 receptor and cell fusion. By contrast, the use of an antibody not directed against rdb (6A2B2 or IFIIB10) does not adversely affect the binding of the envelope and the fusion of the cells significantly, as may be observed by comparing to the results obtained with the anti-HIV 23A5 control antibody. Example 6: Study of the in vivo interaction between the envelope protein and the hASCT2 receptor and the in vivo formation of syncytia To verify the results obtained in vitro, an animal model was designed. Rhabdomyosarcoma cells TelCeB6 (ATCC CRL8 805), in culture in DMEM medium (Gibco Invitrogen 41966-029) supplemented with South American serum, were respectively transfected, with the aid of the LipofectAMINE PLUS™ kit (Gibco Invitrogen), with the DNA corresponding to the HERV-W env gene cloned in the sense orientation at the concentration of 2 µg/µl REPLACEMENT SHEET (RULE 26) (DNA 409), with the DNA corresponding to the HERV-W env gene cloned in the anti-sense orientation at the concentration of 1.5 µg/µl (DNA 410) and with the DNA corresponding to a mutated HERV-W env gene at the concentration of 1.3 µg/µl (DNA LQMV) according to the protocol detailed below. The cells transfected with the DNA 409 are capable of expressing the fusogenic W envelope protein, the cells transfected with the DNA 410 are not capable of expressing the envelope protein and the cells transfected with the DNA LQMV are capable of expressing a nonfusogenic mutated W envelope protein. 1). Protocol: 1st Day: Culture of the TelCeB6 cells: - inoculation of the dishes 100 mm in diameter —> 50- 70% confluence; - incubation in supplemented DMEM medium (6 ml per dish) for 24 hours at 37°C under 5% CO2. 2nd Day: Transfection with the LipofectAMINE PLUSTM kit: 1 Precomplexing of the DNA - mixing of 750 µ1 of medium not supplemented with the abovementioned DNAs in a 15 ml falcon tube (reference 2096), that is 2 µ1 of 409 or 3 µ1 of 410 or 3 µl LQMV; - stirring under vortex of PLUS reagent and adding 20 µ1 thereof to the DNA solution; - stirring under vortex immediately 10 seconds at 1400 rpm; - incubation 15 minutes at room temperature. 5 2 Preparation of the cells - replacement with 5 ml of nonsupplemented medium. REPLACEMENT SHEET (RULE 26) 3 Dilution of the LipofectAMINE In a tube for a dish, mix 30 µ1 of LipofectAMINE Reagent with 750 µ1 of nonsupplemented medium. 4 Complexing of the DMA Mixing of 780 µ1 of dilute LipofectAMINE and 772 µ1 of solution of precomplexed DNA (total: 1552 µ1) ; stirring under vortex immediately 10 sec at 1400 rpm, incubation 15 minutes at room temperature. 5 Transfection and production of recipient animals transplanted with the target cells, treated or not treated by injection of anti-Env antibody Deposition of 1552 µ1 in a dish; incubation 2-3 hours at 37°C under 5% CO2; replacement of the transfection medium with 6 ml of supplemented medium; incubation 1 hour at 37°C under 5% CO2; injection by the intraperitoneal route (IP) to SCID mice (in a volume of 1 ml) , l/5th of each dish at 70% confluence, with or without additional injection of anti-Env protein antibody (monoclonal antibody 2H1H8, polyclonal antibody 69 (anti-SU) and 71 (anti-TM) at 1/100). Production of animals tolerating the transplant and allowing dissemination of the transplanted cells in the body, in parallel with the establishment of pseudo- ascites in the peritoneal cavity. 3rd Day: Collection of the cells from each animal by peritoneal lavage: injection of 2 ml of air followed by 2 ml of physiological saline and then massaging and recovering REPLACEMENT SHEET (RULE 26) the 2 ml of peritoneal fluid (protocol developed for the recovery, in transplanted animals, of the cells implanted in the peritoneal cavity); observation under an "inverted phase" microscope with counting of the syncytia and/or after staining on a slide; immediate reading performed after spreading on slides with a gridded chamber in the presence of Trypan blue (exclusion of the dead cells) . The number of cells which have fused to each other was counted per field with a "wide angle" lens (40) which makes it possible ... to establish the count on more than about one hundred cells so as to have a series of statistically representative counts. A cellular aliquot of each sample is fixed in the presence of methanol/acetone (v/v) and then stored at -20°C until a crystal violet staining is obtained (1%). Photographs were taken of the stained slides. 2) Mice: Groups of 2 mice are inoculated with: the cells transfected with the three types of plasmid (DNAs 409, 410 and LQMV) with no antibody (3x2= 6 mice) the cells transfected with the three types of plasmid (DNAs 409, 410 and LQMV) with the monoclonal antibody 2H1H8 (3x2= 6 mice). 3) Results: The number of fused cells determined by direct reading on a gridded counting chamber per 100 cells is indicated in Table 2 below: Table 2 ECP (Tryptan blue) reading: direct reading of the REPLACEMENT SHEET (RULE 26) Each number represents the number of fused and visualized cells per field studied. As some cells may be superposed in the optical path, the count for the cells appearing fused in the controls is greater than zero. The reality of the syncytia and the discrimination with stacks of cells were then verified by staining the cells on a slide, with visualization of multiple cell nuclei contained in a space delimited by the continuation of a single and sole cell membrane. Moreover, photos showing cells in the course of fusion made it possible to objectify the reality of the fusion upon analysis by phase contrast microscopy and the total absence of an equivalent phenomenon in the controls. To statistically objectify the primary analysis represented by the numbers indicated in Table 2, a Chi-2 test was performed in order to compare the data in Table 2. The results of the statistical analysis taking into account the "background noise" of the primary reading, without secondary analysis after staining on a slide or a search for typical cells in the course of fusion which are never seen in the controls, are the following: REPLACEMENT SHEET (RULE 26) i) Statistical validation of the specificity of the pathogenic effect in vivo: Env expressed (409): 20.5 positives counted on average out of 100 cells, anti-sense Env (410): 9.5 positives counted on average out of 100 cells, mutated Env (LQMV): 6.5 positives counted on average out of 100 cells, mean of the controls (410 and LQMV): 9.5 + 6.5/2 = 8%. Env versus control 410: Chi-2 = 5.89 (p Env versus control LQMV: Chi-2 =9.83 (p Env versus the two controls (410 and LQMV): Chi-2 = 7.69 (p Control 410 versus control. LQMV: Chi-2 =0.61 (difference not significant). The controls are therefore statistically equivalent and there is no "real" difference linked to the type of control. The results obtained from this stage of analysis (not excluding the background noise linked to the artefactual images and by comparing the two types of control with each other (which prove to be equivalent)) was statistically very significant (overall p ii) Statistical validation of the therapeutic activity of the antibodies tested on the pathogenic effect in vivo: Env expressed (409): 20.5 positives counted on average out of 100 cells, REPLACEMENT SHEET (RULE 26) Env expressed (409) + monoclonal antibody 2H1H8: 3.5 positives counted on average out of 100 cells. Env alone versus injection of the antibody 2H1H8: Chi-2 = 15.38 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of result due to chance (p) less than 0.001). Subsequent analyses, by staining, of the specificity of the effects merely confirm the specificity of the effect obtained in vivo in the presence of the Env protein and of antibody, thereby validating the therapeutic effect on the animal model. Example 7: Study in vivo of the binding of the HERV-W Env protein to cells possessing or not possessing type 1 or 2 hASCT receptors and of the inhibition of this binding by injection of antibodies directed against HERV-W Env 1) Materials Soluble protein: supernatant filtered on 0.4 5 µm containing the soluble protein (293T cells transfected with the. plasmid 460 (envelope-spacer-His6) . Expression verified by Western blotting with an anti- RGS-His antibody. Antibody: monoclonal antibody 2H1H8 (IgG, 5.50 mg/ml). Cells: XChASCT2, cellular clone XC (ATCC CCL-165, rat cells) expressing the hASCT2 receptor. DMEM medium (Gibco Invitrogen 41966-029) with South American serum. Preincubation, incubation, labeling in a 1.5 ml Eppendorf tube. 2) Protocol REPLACEMENT SHEET (RULE 2 6) 1 IP (intraperitoneal) inoculation of the XChASCTl, XChASCT2 cells and control cells XChASCT - into SCID mice Injection into mice of l/5th of the flask at 70% confluence in a volume of 2 ml. 2 Preincubation Incubation of the soluble protein supernatant (filtered supernatant of the 293T line) with the monoclonal antibody 2H1H8 (990 µ1 of supernatant with 10 µ1 of antibody (1/lOOth dilution)) for 1 hour at 37°C in the cell incubator with occasional stirring (every 15 minutes). 3 Inoculation IP (intraperitoneal) inoculation of the proteins alone or with the antibody into mice transplanted with the cells (1 X 106 cells per point, that is l/5th of a confluent dish 100 mm in diameter). After injection of the antibody (200 microliters), maintained as IP, for 6 hours, with an occasional peritoneal massage (every 30-60 minutes). 4 Recovery of the cells by peritoneal lavage of the transplanted mice - Centrifugation 3000 revolutions for 5 minutes at + 4°C. - Recovery of the cellular pellet and dilution in the labeling media (maintained at +4°C until fixing). 5 Labeling - Primary antibody: The pellet is taken up in 100 µ1 of anti-RGS His antibody (100th dilution - Quiagen) in a PBA buffer (PBS with 2% fetal calf serum and 0.1% sodium azide) , maintained at +4°C. 1 hour in ice with occasional stirring (every 15 minutes) . REPLACEMENT SHEET (RULE 2 6) Washing in PBA buffer (1 ml per tube), maintained at + 4°C. - Secondary antibody: Centrifugation 3000 revolutions for 5 minutes at +4°C. The pellet is taken up in 100 µl of anti-mouse antibody-FITC (l/20th dilution - DAKO, reference F0479 in a PBA buffer) maintained at +4°C. 1 hour in ice with occasional stirring (every 15 minutes). 2 washes in PBA buffer (1 ml per tube), maintained at + 4°C. Pellet taken up in 500 ]il of PBA, maintained at'+4°C, and analyzed by FACS. Alternatively, analysis by IF after fixing on a slide in acetone/methanol (50%/50%) at -20°C and counter-staining with Evans blue. 3) Mice: Groups of 2 mice are inoculated with: each type of cell (expressing the 2 types of receptor hASCTl and hASCT2 and one not expressing the receptor hASCT - as a control) with no antibody (3x2 = 6 mice) the three types of cell with the Env protein and the monoclonal antibody 2H1H8 (3x2 = 6 mice). 4) Results The results by immunofluorescence (IF) reading with a microscope are presented in Table 3 below: Each number represents the number of cells visualized as fluorescent per field studied. As some cells may have bound fluorescence in a non-specific manner, the count for the cells appearing fluorescent under the control conditions is therefore greater than zero in one of the two fields counted (mean of the two fields = 1/28, that is 0.036%, which is entirely reasonable for the background noise of such a reading technique). The reality of the cells that bound the Env protein to their hASCTl or hASCT2 receptor was then verified by cytofluorometric analysis. In order to statistically objectify the analysis presented in Table 3, a Chi-2 test was performed in order to compare the data obtained under the conditions according to which (i) the Env protein can bind to a receptor hASCTl (control hASCTl) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the grafted control cells which have no receptor (control X) to which the Env protein injected into the corresponding animals cannot bind and thus does not give membrane fluorescence in the presence of an anti-Env antibody and (ii) the Env protein can bind to a receptor hASCTl (control hASCTl) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the injection of a monoclonal antibody directed against Env-SU. The results of the statistical analysis are presented below: i) Statistical validation of the specificity of the pathogenic effect in vivo: REPLACEMENT SHEET (RULE 26) control hASCTl: 24 positives counted on average out of 77 cells, control hASCT2: 23 positives counted on average out of 57 cells, hASCT- cells: 1 positive counted on average out of 28 cells. Env+grafts hASCTl versus hASCT-: Chi-2 = 8.62 (p The cells expressing the hASCTl or hASCT2 receptors at their surface are therefore indeed statistically equivalent and there is no difference in the Env binding to the receptor linked to subtype 1 or 2, under the conditions of the experiment. The results obtained with the animals transplanted with the cells expressing the membrane receptors hASCTl or hASCT2 are statistically significant in the light of the results obtained with the control animals trans¬planted with the cells expressing none of these receptors at their surface. These results confirm the specificity of the effect obtained in vivo in the presence of the Env protein in the animal models. ii) Statistical validation of the therapeutic activity of the antibodies tested on the pathogenic effect in vivo: Env+grafts control hASCTl: 24 positives counted on average out of 77 cells Env+grafts hASCTl + monoclonal antibody 2H1H8: 2 positives counted on average out of 73 cells. Env+grafts hASCTl alone versus injection antibody REPLACEMENT SHEET (RULE 26) 2H1H8: Chi-2 = 21.14 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) less than 0.001). Env+grafts hASCT2: 23 positives counted on average out of 57 cells Env+grafts hASCT2 + monoclonal antibody 2H1H8: 1 positive counted on average out of 45 cells. Env+grafts hASCT2 alone versus injection antibody 2H1H8: Chi-2 = 20.31 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) overall less than 0.01). The validation of the animal models against the appropriate controls makes it possible to demonstrate that antibodies may have a therapeutic activity by significantly inhibiting the pathogenic effects of the ~HERV-W Env protein. Example 8: Alignment of the sequences of the interference group The protein sequences of the envelopes of the retroviruses HERV-W (swiss-prot Q9UQF0), RD114 (swiss-prot Q98654), REV (swiss-prot P31796), BAEV (swiss-prot P10269), SRVl (swiss-prot P04027), SRV2 (swiss-prot P51515) and MPMV (swiss-prot P07575) were aligned with the aid of the MacVector software with the ClustalW procedure. The signal peptide, the SU (surface unit) subunit and the TM (Trans membrane) subunit are indicated. The receptor binding site is underlined. REPLACEMENT SHEET (RULE 26) CLAIMS 1. A peptide domain necessary for the interaction between the envelope of a virus belonging to the HERV-W pterference group and an hASCT receptor, defined in hat it starts with an N-terminus and ends with a -terminus, and in that: he N-terminus is defined by a motif chosen from SEQ ID 1 to SEQ ID No. 29, he C-terminus is defined by a motif chosen from SEQ No. 30 to SEQ ID No. 40, and in that said peptide domain comprises, between the -terminus and the C-terminus, at least one motif hosen from SEQ ID No. 41, SEQ ID No. 42 and SEQ ID . 73. A nucleotide sequence encoding a peptide domain as fined in claim 1. An epitope derived from the peptide domain as ifined in claim 1, characterized in that it induces an amune response against a virus belonging to the HERV-W iterference group. A nucleotide sequence encoding an epitope as ifined in claim 3. An expression vector, characterized in that it emprises a nucleotide sequence as claimed in either of aims 2 and 4, and the means necessary for its pression. A microorganism or host cell transformed with at east one expression vector as defined in claim 5. An antibody directed against a peptide domain as defined in claim 1 or against an epitope as defined in claim 3. 8. The use of at least one peptide domain as defined in claim 1 or of at least one epitope as defined in claim 3, or of at least one antibody as defined in claim 7 or of at least one nucleotide sequence as defined in claims 2 or 4 placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, for the preparation of a medicament intended for the inhibition, prevention or treatment of an infection caused by a virus belonging to the HERV-W interference group in an animal, preferably humans. 9. A pharmaceutical composition comprising, by way of active substance, at least one peptide domain as defined in claim 1, or at least one epitope as defined in claim 3, or alternatively at least one of the nucleotide sequences as defined in claims 2 or 4, placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, or alternatively at least one antibody as defined in claim 7 in combination with a pharmaceutically appropriate vehicle. 10. A diagnostic composition for the detection and/or quantification of a virus belonging to the HERV-W interference group and/or the quantification of an immune response against a virus belonging to the HERV-W interference group, comprising at least one peptide domain as defined in claim 1, at least one epitope as defined in claim 3, or at least one of the nucleotide sequences as defined in claims 2 or 4, or at least one antibody as defined in claim 7. 11. A method for the detection and/or quantification of a virus belonging to the HERV-W interference group REPLACEMENT SHEET (RULE 26) in a biological sample taken from an individual liable to be infected by said virus, characterized in that it comprises the steps consisting in: bringing said biological sample into contact with at least one antibody as defined in claim 7 under conditions allowing the formation of a complex between the virus and the antibody, and detecting and/or quantifying the formation of said complex by any appropriate means. 12. The use of the composition as claimed in claim 10 for the in vitro screening of a virus belonging to the HERV-W interference group in a biological sample or specimen. 13. The use of a peptide domain as claimed in claim 1 or of an epitope as defined in claim 3, or of an antibody as defined in claim 7, for inhibiting the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor. 14. The use of a peptide domain as claimed in claim 1, for identifying chemical or biological molecules whose interaction with all or part of this peptide domain blocks the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor. 15. The use of a peptide domain as claimed in claim 1 for generating antibodies blocking the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor. 16. A method for determining a polypeptide region necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, characterized in that: the nucleotide and/or peptide sequence of the REPLACEMENT SHEET (RULE 2 6) precursor envelope of said virus is identified the signal part is excluded a serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic acid-Xf-Xg motif is detected, which corresponds to SEQ ID No. 4 3 in which Xa, Xb, Xc, Xd, Xg, Xf, Xg are any amino acids the C-terminus is excluded between 15 and 25 amino acids after said serine-aspartic acid-Xg- Xb-Xc-Xd-Xg-aspartic acid-Xf-Xg motif, which corresponds to SEQ ID No. 43. 17. A peptide domain which can be obtained by the method as claimed in claim 16. REPLACEMENT SHEET (RULE 26)

Full Text

Peptide domain required for interaction between the
envelope of a virus pertaining to the HERV-W
interference group and an hASCT receptor
The present invention relates to a polypeptide domain responsible for interactions between a retroviral envelope of the HERV-W interference group and the receptors of the hASCT family.
Human endogenous retroviruses (HERVs) constitute 8% of the human genome and are involved both in pathologies and in nonpathological phenomena.
The human endogenous retrovirus family W (HERV-W) is derived from an infectious retroviral element that was integrated into the germ line 25 to 40 million years ago. the_HERV-W envelope protein, also called syncytin, is a fusogenic glycoprotein involved in the formation of the syncytiotrophoblastic layer of the placenta. It is encoded by the env gene of the proviral locus ERVWl and synthesized in the form of a gPr7 3 precursor which is specifically cleaved into two mature proteins, a surface subunit gp50 (SU) and a transmembrane subunit gp24 (TM).
.In vitro, syncytin of the HERV-W- family induces a cell to cell fusion that is dependent on its interaction with a receptor-transporter of amino acids of the ASCT family (h-ASCT2, hASCTl). Phylogenic studies then showed that syncytin is related to a group of retroviruses comprising in particular the cat endogenous virus RD114, the monkey endogenous virus BaEV, simian retroviruses and avian retroviruses: avian reticuloendotheliosis virus REV-A and spleen necrosis virus SNV, all having in common the type 2 sodium-dependant neutral amino acid receptor-transporter or
REPLACEMENT SHEET (RULE 26)

ASC12 (rasko ex. al, 1999, Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2129-2134; Tailor et al, 1999 Journal of Virology, vol. 73(5) May 1999, P. 4470-4474). Thus, the infection of cells with viruses of this retrovirus group leads to a specific reduction in the transport of amino acids (Rasko et al., 1999). The infection of a cell with one of these retroviruses (or the expression of one of these envelopes in the cell) prevents, through interference (interaction) in relation to a receptor of the ASCT family, the infection of this same cell by another of these retroviruses or the fusion with another cell expressing another envelope. Through interference in relation to a receptor of the ASCT family, the infection of a cell by one of these retro¬viruses prevents the infection by another of these retroviruses. All these retroviruses belong to the same HERV-W virus interference group.
The mechanisms of binding between the envelope and the ASCT receptor remain obscure, and to date no domain for binding to an ASCT receptor has been identified and defined either in the SU of the HERV-W envelope protein or in the SUs of retroviruses of the same interference group. This theme is nevertheless essential since the inhibition of the envelope/ASCT receptor interaction would in addition make it possible to prevent the entry of a retrovirus into the cell, and therefore to block its replication cycle, to block the phenomenon of envelope/ASCT receptor interaction and/or of cell fusion which may be involved in the formation of tumors, in the proliferation of metastasic cells or in drug resistance phenomena (see by way of illustration the publication "Cell fusion: A hidden enemy?. Cancer Cell: May 2003, vol. 3), to block the phenomenon of envelope/ASCT receptor interaction and/or of cell fusion which may be involved in nervous system diseases and even to inhibit the cell-cell fusion involved in trophoblastic differentiation (contraceptive

vaccination). Furthermore, the inhibition of the envelope/hASCT receptor interaction could prevent tumor propagation by counteracting a local immunosuppression which may result from the envelope/hASCT receptor interaction. Indeed, it has been shown, on the one hand, that the infection of cells with viruses of this retrovirus group (in particular those inducing immunodeficiencies) leads to a specific reduction in the transport of amino acids (Rasko et al, PNAS, vol. 96. pp 2129-2134 (1999)), and on the other hand, a direct link is proposed between the impairment of the transport of amino acids and immunosuppression (Espinosa A, Villarreal LP., T-Ag inhibits implantation by EC cell derived embryoid bodies. Virus Genes. 2000; 20(3): 195-200; JE, Battini JL, Gottschalk RJ, Mazo I, Miller AD., The RD114/simian type D retrovirus receptor is a neutral amino acid transporter. Proc Natl Acad Sci USA, 1999, Mar 2; 96(5): 2129-34). Thus, as regards nervous system diseases, it is known that the hASCT receptors are involved in the specific transport of neutral amino acids and that neuronal cells, for the transmission of information, predominantly use neuromediators of a polypeptide nature. Thus, the binding of the Env-HERV-W protein to receptors which normally have to transport the amino acids required for the synthesis of neuromediators can affect the capacity of the neurons to synthesize the neuromediators by reducing the entry of the physiological agonists such as amino acids via the ASCT receptors. Moreover, if neurons whose intercellular networks form connections which are essential for the transmission of information circulating in the brain and the spinal cord, form syncytia following a fusion of several neurons which is induced by the Env-HERV-W protein, all the networks for transmission of information become disrupted and connected to the same fused "cellular package" and, furthermore, the neuromediator production activity of each cell is no longer individualized or connected to
REPLACEMENT SHEET (RULE 26)

the upstream or downstream conduction pathways (dendrites and axons) which are specific to it.
Surprisingly, the inventors have identified the polypeptide region responsible for the interactions between the envelope of a virus, belonging to the HERV-W interference group and an hASCT receptor.
To this effect, the present invention relates to a peptide domain necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, defined in that it starts with an N-terminus and ends with a C-terminus, and in that:
■ the N-terminus is defined by a motif,
consisting of the amino acids (Z)a-proline-
cysteine-X-cysteine
in which
Z is any amino acid
a is an integer between 2 and 30
X is any amino acid,
said motif being chosen from SEQ ID No. 1 to
SEQ ID No. 29 and SEQ ID No. 44 to SEQ ID
No. 72
the C-terminus is defined by a motif consisting
of the amino acids serine-aspartic acid-Xg-Xb-
Xc-Xd-Xe aspartic acid-Xf-Xg-(Z) p
in which
Xa, Xb, Xc, Xd, Xe, Xf, Xg are any amino acids
Z is any amino acid
P is an integer between 15 and 25, preferably
20
said motif being chosen from SEQ ID No. 30 to
SEQ ID No. 40, and in that said peptide domain comprises, between the N-terminus and the C-terminus, at least one motif chosen from the following motifs:
REPLACEMENT SHEET (RULE 26)

a motif consisting of the amino acids cysteine-
tyrosine-X2-X3-X4-X5-X6- cysteine,
in which X2, X3, X4, X5, Xg are any amino acids said motif corresponding to SEQ ID No. 41
a motif consisting of the amino acids cysteine-
X7-X8-X9-X10-X11-X12-X13-X14-X15-cysteine-
tryptophan,
in which X7, X8, X9, X10, X11, X12, X13, X14, X15 are any amino acids
said motif corresponding to SEQ ID No. 42 or SEQ ID No. 73.
The expression peptide domain according to the invention is understood to mean a minimum region of the envelope of a virus of the HERV-W interference group necessary for the recognition of an hASCT receptor.
The peptide domains of the invention may be obtained by the genetic engineering technique which comprises the steps of:
culturing a microorganism or eukaryotic cells transformed with the aid of a nucleotide sequence according to the invention, and recovering the peptide domain produced by said microorganism or said eukaryotic cells.
This technique is well known to a person skilled in the art. For further details concerning it, reference may be made to the book below: Recombinant DNA Technology I, Editors Ales Prokop, Raskesh K Bajpai; Annals of the New York Academy of Sciences, Volume 646, 1991. The peptide domains of the invention may also be prepared by conventional peptide syntheses well known to a person skilled in the art.
The expression interference group is understood to mean all the viruses for which the infection (expression) of a cell by one of its members prevents infection by '
REPLACEMENT SHEET (RULE 26)

another member of the group by receptor interference.
The expression any amino acid is understood to mean in particular an amino acid chosen from arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, asparagine, threonine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, cysteine, glycine, proline.
The expression hASCT receptor is understood to mean any sodium-dependent neutral amino acid transporter.
The expression motifs is understood to mean a succession of amino acids corresponding to a particular region of interest of the peptide domain according to the invention, which is expressed by all the viruses of the HERV-W virus interference group.
Preferably, a is an integer between 3 and 18.
Preferably X or Xaa of the Pro Cys Xaa Cys motif in SEQ ID No. 1 to SEQ ID No. 29 is an amino acid chosen from aspartic acid, glutamic acid, arginine: these sequences are those preferably chosen from SEQ ID No. 44 to SEQ ID No. 72.
Preferably, Xa, Xb, Xc or the amino acids at positions 3, 4 and 5 of SEQ ID Nos. 30 to 40 are a glycine, Xa or the amino acid Xaa at position 6 of SEQ ID Nos. 30 to 4 0 is an amino acid chosen from proline and valine; Xe or the amino acid Xaa at position 7 of SEQ ID Nos. 30 to 40 is an amino acid chosen from glutamine, leucine and threonine; Xf or the amino acid Xaa at position 9 of SEQ ID Nos. 30 to 40 is an amino acid chosen from lysine, threonine, methionine and glutamine, Xg or the amino acid at position 10 of SEQ ID Nos. 30 to 40 is an amino acid chosen from alanine, lysine, isoleucine,
REPLACEMENT SHEET (RULE 26)

threonine and valine.
Preferably, [3 is an integer equal to 20.
Preferably, X2 or the amino acid Xaa at position 3 of SEQ ID No. 41 is an amino acid chosen from asparagine, threonine, glutamic acid, histidine, X3 or the amino acid Xaa at position 4 of SEQ ID No. 41 is an amino acid chosen from histidine, alanine, serine, lysine, glutamic acid; X4 or the amino acid Xaa at position 5 of SEQ ID No. 41 is an amino acid chosen from tyrosine, threonine, alanine, X5 or the amino acid Xaa at position 6 of SEQ ID No. 41 is an amino acid chosen from glutamine, arginine, threonine, Xe or the amino acid Xaa at position 7 of SEQ ID No. 41 is an amino acid chosen from leucine, glutamine, glutamic acid.
Preferably X7 or the amino acid Xaa at position 2 of SEQ ID No. 42 is an amino acid chosen from proline, chreonine, arginine and asparagine; Xg or the amino acid Xaa at position 3 of SEQ ID No. 42 is an amino acid chosen from glycine, glutamic acid, asparagine, Xg 3r the amino acid Xaa at position 4 of SEQ ID No. 42 is an amino acid chosen from glycine, asparagine, soleucine, threonine, serine, Xio or the amino acid Xaa it position 5 of SEQ ID No. 42 is lysine or is deleted;
k11 or the amino acid Xaa at position 6 of SEQ ID No. 42 ss an amino acid chosen from lysine, valine, soleucine, leucine, X12 or the amino acid Xaa at position 7 of SEQ ID No. 42 is an amino acid chosen ;rom glycine, asparagine; X13 or the amino acid Xaa at position 8 of SEQ ID No. 42 is an amino acid chosen from glutamine, lysine, valine, X14 or the amino acid kaa at position 9 of SEQ ID No. 42 is an amino acid thosen from valine, proline, serine, threonine, X15 or the amino acid Xaa at position 10 of SEQ ID No. 42 is in amino acid chosen from valine, isoleucine.
REPLACEMENT SHEET (RULE 26)

Preferably,
the amino acid Xaa at position 2 of SEQ ID No. 73 is
chosen from proline, threonine, arginine and
asparagine,
the amino acid Xaa at position 3 of SEQ ID No. 73 is
chosen from glycine, glutamic acid, asparagine,
the amino acid Xaa at position 4 of SEQ ID No. 73 is
chosen from glycine, asparagine, isoleucine, threonine,
serine,
the amino acid Xaa at position 5 of SEQ ID No. 73 is
chosen from lysine, valine, isoleucine, leucine,
the amino acid Xaa at position 6 of SEQ ID No. 73 is
chosen from glycine, asparagine,
the amino acid Xaa at position 7 of SEQ ID No. 73 is
chosen from glutamine, lysine, valine,
the amino acid Xaa at position 8 of SEQ ID No. 73 is
chosen from valine, proline, serine, threonine,
the amino acid Xaa at position 9 of SEQ ID No. 73 is
chosen from valine, isoleucine.
Deletions are possible in the domains according to the invention indicated above. According to a particular embodiment of the invention, X10 is deleted.
The invention also relates to a nucleotide sequence encoding a peptide domain according to the invention above.
Such sequences may be prepared by chemical synthesis and genetic engineering using techniques well known to a person skilled in the art and described, for example, in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 198 9.
The invention also relates to an epitope derived from the peptide domain according to the invention, characterized in that it induces an immune response against a virus belonging to the HERV-W interference
REPLACEMENT SHEET (RULE 2 6)

group.
The expression epitope is understood to mean all or part of the peptide domain according to the invention recognized by a receptor located at the surface of a B or T lymphocyte or of a circulating antibody.
The expression immune response is understood to mean all the biological mechanisms which allow . a pluricellular organism to maintain the coherence of the cells and tissues which constitute it and to ensure its integrity -in response to any attack which modifies the molecular structures of its constituents or which introduces foreign molecules into the organism.
The invention also relates to a nucleotide sequence encoding an epitope as defined above. As indicated above, such sequences may be prepared by chemical synthesis and genetic engineering using techniques well known to a person skilled in the art and described, for example, in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 1989.
The invention also relates to an expression vector characterized in that it comprises a nucleotide sequence according to the invention, and the means necessary for its expression.
By way of expression vector, there may be mentioned, for example, plasmids, viral vectors of the vaccinia virus, adenovirus, baculovirus, poxvirus or retrovirus type, bacterial vectors of the salmonella or BCG type.
The expression means necessary for its expression is understood to mean any means which make it possible to obtain a peptide from a nucleotide sequence, such as in particular a promoter, a transcription terminator, a replication origin and preferably a selectable marker.
REPLACEMENT SHEET (RULE 26)

The vectors of the invention may also comprise sequences necessary for targeting peptides to particular cell compartments.
The invention also relates to a host microorganism or cell transformed with at least one expression vector according to the invention.
By way of examples of microorganisms which are suitable
for the purposes of the invention, there may be
mentioned yeasts, such as those of the following
families: Saccharomyces, Schizosaccharoyces,
Kluveromyces, Pichia, Hanseluna, Yarowia,
Schwaniomyces, Zygosaccharomyces; Saccharomyces
cerevisiae, Saccharomyces carlshergensis and
Kluveromyces lactis being preferred; and bacteria such as E.' coli and those of the following families: Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus _a.ndi Streptomyces.
By way of examples of transformed host cells, there may be mentioned cells derived from animals such as mammals, reptiles, insects and the like. The preferred eukaryotic cells are cells derived from the Chinese hamster (CHO cells), from monkeys (COS and Vero cells), from young hamster kidney (BHK cells), from pig kidney (PK 15 cells) and from rabbit kidney (RK13 cells), human osteosarcoma cell lines (143 B cells), human HeLa cell lines and human hepatoma cell lines (of the Hep G2 cell type), and insect cell lines (for example Spodoptera frugiperda), a human embryonic kidney cell line (for example HEK293T). The host cells may be provided in cultures in suspension or in flasks, in tissue cultures, organ cultures and the like.
The invention also relates to an antibody directed against a peptide domain according to the invention or
REPLACEMENT SHEET (RULE 2 6)

against an epitope according to the invention.
The expression antibody is understood to mean both a whole antibody and an antibody fragment.
The recombinant antibodies may be obtained according to conventional methods known to a person skilled in the art, from prokaryotic organisms, such as bacteria, or from eukaryotic organisms, such as yeasts, mammalian, plant, insect or animal cells, or by extracellular production systems.
The monoclonal antibodies may be prepared according to conventional techniques known to a person skilled in the art such as the hybridoma technique whose general principle is recalled below.
In a first instance, an animal, generally a mouse (or cultured cells in the context of in vitro immunizations) , is immunized with a target antigen of interest, whose B lymphocytes are then capable of producing antibodies against said antigen. These antibody-producing lymphocytes are then fused with "immortal" myelomatous cells (murine in the example) in order to give rise to hybridomas. From the heterogenous mixture of cells thus obtained, a selection of the cells capable of producing a particular antibody and of multiplying it indefinitely is then carried out. Each hybridoma is multiplied in clone form, each leading to the production of a monoclonal antibody whose recognition properties in relation to the antigen of interest may be tested, for example by ELISA, by one-or two-dimensional immunotransfer, by immuno¬fluorescence or with the aid of a biosensor. The monoclonal antibodies thus selected are subsequently purified in particular according to the affinity chromatography technique.
REPLACEMENT SHEET (RULE 26)

The expression antibody fragment is understood to mean any antibody fragment following an immune response against a virus belonging to the HERV-W interference group. These antibody fragments may, for example, be obtained by proteolysis. Thus, they may be obtained by enzymatic digestion, resulting in fragments of the Fab type (treatment with papain; Porter RR, 1959, Biochem. J., 73: 199-125) or of the F(ab)'2 type (treatment with pepsin; Nisonoff A. et al., 1960, Science, 132: 1770-1771) . They may also be prepared by the recombinant route (Skerra A., 1993, Curr. Opin. Immunol., 5: 256-262). Another antibody fragment which is suitable for the proposals of the invention comprises an Fv fragment which is a dimer consisting of the noncovalent combination of the variable light (VL) domain and of the variable heavy (VH) domain of the Fab fragment, and therefore the combination of two polypeptide chains. In order to improve the stability of the Fv fragment due to the dissociation of the two polypeptide chains, this Fv fragment may be modified by genetic engineering by inserting a suitable linker peptide between the VL domain and the VH domain (Huston P. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5879-5883). The expression scFv fragment ("single chain Fragment variable") is then used because it consists of a single polypeptide chain. The use of a linker peptide preferably composed of 15 to 25 amino acids makes it possible to link the C-terminus of one domain to the N-terminus of the other domain, thus constituting a monomeric molecule endowed with binding properties similar to those of the antibody in its complete form. Both orientations of the VL and VH domains are suitable (VL-linker-VH and VH-linker-VL) because they exhibit identical functional properties. Of course, any fragment known to a person skilled in the art and exhibiting the immunological characteristics defined above are suitable for the purposes of the invention.
REPLACEMENT SHEET (RULE 2 6)

The invention also relates to the use of at least one peptide domain according to the invention, of at least one epitope according to the invention, of at least one antibody according to the invention or of at least one nucleotide sequence according to the invention, for the preparation of a medicament intended for the inhibition, prevention or treatment of an infection caused by a virus belonging to the HERV-W interference group in an animal, preferably humans. The peptide domain according to the invention may be used in particular for targeting cells expressing a receptor of the hASCT family in order to transduce a signal, and modulate the flow of amino acids (cancer treatments) .
The expression elements necessary for a constitutive expression of peptides is understood to mean a ubiquitous promoter specific to eukaryotic cells. By way of elements necessary for an inducible expression of the peptides, there may be mentioned the elements for regulating the E. coli operon for resistance to tetracycline (Gossen M. et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992)).
The use of at least one peptide domain according to the invention, of at least one epitope according to the invention, or of at least one nucleotide sequence according to the invention is particularly suitable for the preparation of a medicament intended for the prevention of an infection caused by a virus belonging
I to the HERV-W interference group in an animal, preferably humans. The use of at least one antibody according to the invention is particularly suitable for the preparation of a medicament intended for the inhibition or treatment of an infection or a pathology
induced by a virus belonging to the HERV-W interference group in an animal, preferably humans.
The invention also relates to a pharmaceutical REPLACEMENT SHEET (RULE 26)

composition comprising, by way of active substance, at least one peptide domain according to the invention, at least one epitope according to the invention, or alternatively at least one of the nucleotide sequences according to the invention, in particular placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, in combination with a pharmaceutically appropriate vehicle. The invention also relates to a pharmaceutical composition comprising, by way of active substance, at least one antibody according to the invention, in combination with a pharmaceutically appropriate vehicle.
Of course, persons skilled in the art will easily determine the pharmaceutically appropriate vehicle and the quantity of peptide domains, of epitopes, of nucleotide acids or of antibodies to be used according to the constituents of the pharmaceutical composition.
In the pharmaceutical compositions according to the invention, for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, rectal or transdermal administration, the active substance may be administered in unit forms for administration or as a mixture with conventional pharmaceutical supports and intended for administration by the oral route, for example in the form of a tablet, a gelatin capsule, an oral solution, and the like, or by the rectal route, in the form of a suppository, or by the parentral route, in particular in the form of a solution for injection, in particular by the intravenous, intradermal or subcutaneous route, and the like, according to conventional protocols well known to persons skilled in the art. For topical application, the active substance may be used in creams, ointments, lotions, eyedrops.
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When a solid composition in tablet form is prepared, the active substance is mixed with the pharmaceutically acceptable excipient, also called a pharmaceutical vehicle, such as gelatin, starch, lactose, magnesium stearate, talc, gum Arabic or the like. The tablets may be coated with sucrose, with a cellulose derivative or with other appropriate materials. It is also possible to treat them such that , they have a prolonged or delayed activity and they continuously release a predetermined quantity of the active substance. It is also possible to obtain a preparation as gelatin capsules by mixing the active substance with a diluent and pouring the mixture into soft or hard gelatin capsules. It is also possible to obtain a preparation in syrup form or for administration in the form of drops, in which the active substance is present together with a sweetener, an antiseptic, such as in particular methylparaben and propylparaben, and a taste enhancer or an appropriate colorant. The powders or water-dispersible granules may contain the active substance in the form of a mixture with dispersing agents or wetting agents, or suspending agents, well known to persons skilled in the art. For parentral administration, aqueous suspensions, isotonic saline solutions or sterile solutions or solutions for injection which contain dispersing agents, pharmacologically compatible wetting agents, such as in particular polyethylene glycol or butylene glycol, are used.
The medicament or the pharmaceutical composition according to the invention may additionally comprise an activating agent which induces the effects of a medication or reinforces or supplements the effects of the principal medication, by increasing in particular the bioavailability of the principal medication.
The dosage depends on the seriousness of the condition REPLACEMENT SHEET (RULE 26)

and will be adapted according to a conventional protocol. As a guide, when the active substance is a monoclonal antibody, the weekly dose is from 1 to 10 mg/kg, in combination with a pharmaceutically acceptable excipient.
The invention also relates to a diagnostic composition for the detection and/or quantification of a virus belonging to the HERV-W interference group, or the detection and/or quantification of an immune response against said virus, comprising at least one peptide domain according to the invention, at least one epitope according to the invention, at least one of the nucleotide sequences according to the invention, or at least one antibody according to the invention.
A diagnostic composition comprising at least one peptide domain according to the invention, at least one epitope according to the invention, at least one of the nucleotide sequences according to the invention, is particularly suitable if it is desired to determine if as apatient has an immune response against a virus belonging to the HERV-W interference group while a diagnostic composition comprising at least one antibody according to the invention is particularly suitable for the detection and/or quantification of a virus belonging to the HERV-W interference group.
The invention also relates to a method for the detection and/or quantification of a virus belonging to the HERV-W interference group in a biological sample taken from an individual liable to be infected by said virus, characterized in that it comprises the steps consisting in:
bringing said biological sample into contact with at least one antibody according to the invention under conditions allowing the formation of a complex between the virus and
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the antibody, and
detecting and/or quantifying the formation of
said complex by any appropriate means.
The expression biological sample is understood to mean a biological sample of human or animal origin liable to contain said virus, such as a sample of blood, plasma, serum, urine, cerebrospinal fluid, or of tissues, such as placenta, testicles, prostate and breast.
The step of bringing into contact is a step that is conventionally known to a person skilled in the art.
The detection/quantification step may be carried out by any detection means known in the field of immunological assays of very small molecules, such as direct detection, that is to say without the intermediary of a binding partner or of binding partners, and indirect detection, that is to say through the intermediary of a binding partner or of binding partners.
The direct detection of the binding between the antibody or antibody fragment of the invention and the virus may be carried out for example by surface plasmon resonance or by cyclic voltammetry on an electrode bearing a conducting polymer. In this case, the antibody of the invention serves to immunocapture all or part of the virus, which is then eluted. The elution may be carried 'out by any elution method known to a person skilled in the art, such as a pH shock.
In the case of indirect detection, the second step of the method of the invention may be carried out according to the conventional ELISA competition assay technique. The antibody of the invention then serves as binding partner serving to capture all or part of the virus in the sample. The detection may then be performed by competition between all or part of the
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virus which may be contained in the sample to be tested and a previously labeled known quantity of virus.
The expression labeling is understood to mean the attachment of a marker capable of directly or indirectly generating a detectable signal. A nonlimiting list of these markers consists of:
■ enzymes which produce a detectable signal, for
example, by colorimetry, fluorescence,
luminescence, such as horseradish peroxidase,
alkaline phosphatase, acetylcholine esterase, P-
galactosidase, glucose-6-phosphate dehydrogenase,
chromophores such as luminescent, coloring
compounds,
radioactive molecules such as 32P, 35S or 1251,
fluorescent molecules such as fluorescein,
rhodomine, alexa or phycocyanins, and
particles such as gold or magnetic latex
particles, liposomes.
ndirect labeling systems may also be used, such as, for example, via another ligand/anti-ligand pair. The igand/anti-ligand pairs are well known to a person killed in the art, and the following pairs may be entioned for example: biotin/streptavidin, biotin/avidin, apten/antibody, antigen/antibody, peptide/antibody, agar/lectin, polynucleotide/complementary strand for the plynucleotide. In this case, it is the ligand which is ound to the binding partner. The anti-ligand may be etected directly by the markers described in the roceeding paragraph or may itself be detected by a igand/anti-ligand.
hese indirect systems may lead, under certain anditions, to an amplification of the signal. This ignal amplification technique is well known to a erson skilled in the art, and reference may be made to he previous patent applications FR98/10084 or
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WO95/08000 by the applicant or to the article J. Histochem. Cytochem., (1997), 45: 481-491.
The labeling of molecules is widely known to a person skilled in the art and is described for example by Greg T. Hermanson in Bioconjugate Techniques, 1996, Academic Press Inc., 525B Street, San Diego, CA92101 USA.
Depending on the type of labeling used, such as for
example using an enzyme, a person skilled in the art
will add reagents which allow visualization of the
labeling.
Such reagents are widely known to a person skilled in
the art and are described in particular .in Principles and Practice of Immunoessay, 2nd edition, Edited by C. Price, D.G. Newman, Stockton Press, 1997, 345 Park Avenue South, New York.
The invention also relates to the use of the above
composition for the in vitro screening of a virus
belonging to the HERV-W interference group in a
biological sample or specimen. In particular, the early
screening of a virus such as SRVl and SRV2, viruses
that are involved in immunodeficiency mechanisms in
monkeys, makes it possible to provide a treatment
suitable for the host before the appearance of an
immunodeficiency. Moreover, the early screening of HERV-W, involved in placental pathologies, makes it possible to modulate its expression, for example, during a preeclampsia.
The invention also relates to the use of a peptide domain according to the invention or of an epitope according to the invention, or of an antibody according to the invention, for inhibiting the interaction between the envelope of a virus belonging to the HERV-W
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interference group and an ASCT receptor. This makes it possible, in particular, to obtain a contraceptive immunotherapy.
The invention also relates to the use of a peptide domain according to the invention for identifying chemical or biological molecules whose interaction with all or part of this peptide domain blocks the interaction between the envelope of a virus belonging to the HERV-W interference group and an ASCT receptor. For example, when a peptide domain according to the invention of HERV-W is used, this makes it possible to obtain in particular chemical or biological molecules that are highly suitable in order to obtain a contraceptive treatment. The use of such chemical mole¬cules to inhibit the interaction between the envelope of a virus belonging to the HERV-W interference group and an ASCT receptor is of therapeutic interest.
As a guide, two generic methods allowing the screening of chemical or biological molecules capable of inhibiting the env/receptor interaction are described below.
In a context where it is possible to produce a soluble envelope, it is advisable to determine if a chemical or biological molecule alters the env/receptor interaction according to an ELISA type method using cells expressing at least one hASCT receptor in a capture phase. Thus, on a 96-well plate, cells expressing an hASCT receptor of interest are cultured or adsorbed, and an env/receptor interaction is detected via the use of a labeled soluble envelope (histine tag, GPF fusion), and therefore capable of generating a reference signal that can be assayed. If a signal reduction is observed after preincubation of said soluble envelope with a chemical or biological molecule, that means that the chemical or biological
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molecule alters the env/receptor interaction. Alter¬natively, it is possible to use a retroviral vector pseudotyped by the envelope of interest and expressing a detectable marker (LacZ) and to carry out the same test. It is also possible to select molecules of interest via a measurement of fusion inhibition. Cells expressing the receptor of interest (cell-recept), for example HeLa or XC-RDR cells, and cells constitutively expressing a marker (for example, LacZ) and transiently or stably expressing the envelope of interest (cell-env-LacZ) are used; the envelope of interest was modified beforehand at the level of its intra-cytoplasmic tail by exchange with the intracytoplasmic domain of HERV-W env so as to make it constitutively fusogenic (Cheynet et al, 79(9): 5586-5593, 2005). The bringing into contact of the two cell types, "cell-recept" in excess and "cell-env-LacZ" in insufficient amount, leads to the formation of multinucleated giant cells or syncytia, containing one or two blue nuclei derived from "cell-env-LacZ" and tens of white nuclei derived from "cell-recept". An identical co-culture performed in the presence of a chemical or biological molecule altering the env/receptor interaction leads to a reduction in the number of syncytia and their nucleus content. Automation of such measurements with the aid of a CCD camera is possible.
The invention also relates to the use of a peptide domain in accordance with the invention for generating antibodies blocking . the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor.
The invention also relates to a method for determining a polypeptide region necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, characterized in that:
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the nucleotide and/or peptide sequence of the
precursor envelope of said virus is identified
the signal part is excluded
a serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic
acid-Xf-Xg is detected domain
in which
Xa, Xb, Xc, Xd, Xg, Xf, Xg, are any amino acids
which correspond to SEQ ID No. 43
the C-terminus is excluded between 15 and 25
amino acids, preferably 20 amino acids, after
said serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic
acid-Xf-Xg domain, which corresponds to SEQ ID
No. 43.
Preferably, Xa, Xb, Xc is an amino acid which is glycine, Xd is an amino acid chosen from proline and valine, Xg is an amino acid chosen from glutamine, leucine and threonine, Xf is an amino acid chosen from lysine, threonine, methionine and glutamine, Xg is an amino acid chosen from alanine, lysine, isoleucine, threonine and valine.
For the purposes of the present invention, the
nucleotide sequence and/or peptide consequent of the
precursor envelope of said virus is identified by any
means known to a person skilled in the art, who may
refer in particular to Maniatis (ed. 1989).
The signal part is excluded by any means known to a person skilled in the art, as described in particular in "Improved Prediction of Signal Peptide: SignalP 3.0" Jannick Dyrl0v Bendtsen, Henrik Nielsen, Gunnar von Hiejne and S0ren Brunak, J. Mol. Biol., 340: 783-795, 2004.
Said domain is detected by any means known to a person skilled in the art, that is to say using Blast or Fasta type software (see, in particular, Altschul SF, Gish W,
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Miller W, Myers EW, Lipman DJ., Basic local alignment search tool. J. Mol. Biol. 1990 Oct 5; 215(3): 403-10).
The invention also relates to a peptide domain capable of being obtained by the above method.
The accompanying figures are given by way of explanatory example and have no limiting character. They will make it possible to better understand the invention.
figure 1 illustrates the phenotypical characteristics and properties of soluble recombinant proteins derived from Env-W. In particular, figure la illustrates the Largest soluble recombinant protein comprising all or part of the SU and TM subunits (Env-Gp60) and the soluble recombinant protein corresponding to the SU subunit (EnvSU). Figure lb represents the flow cytometry analysis of the test for binding of the EnvSU recombinant protein to XC hASCT2 and XC hASCTl cells expressing the hASCT2 and hASCTl receptors, respectively. Figure 1c illustrates the test of interference of binding to the TE671 cells (control ASCT2), TE671RD cells (blocked hASCT2) and TE671galv cells (blocked Pitl) .
Figure 2 illustrates the definition of the minimum oinding domain of the ERV-W envelope to the hASCT2 receptor (RBD for receptor binding domain). In particular, figure 2a describes all the deletion mutants designed from EnvSU. Figure 2b represents the flow cytometry analysis of the test of binding of the recombinant proteins derived from EnvSU to the XC hASCT2 cells expressing the hASCT2 receptor, in particular the binding of the Envl97, Envl68 and Envl44 Tiutants and the binding defect of the Env69-317, Envl69-317 and Envll7 mutants.
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Figure 3a illustrates the definition of an immunogenic peptide inside the domain according to the invention (RBD) corresponding to region 21-144 of the precursor of the HERV-W envelope protein. Figure 3b shows the inhibition of the binding of the RBD to its receptor with the aid of an antibody produced from the immunogenic peptide (antiSU-EnvW) and the absence of inhibition of RBD-receptor binding in the presence of a nonspecific antibody (antlTM-EnvW).
Figure 4 represents the alignment of the retroviral envelope sequences belonging to the same interference group and shows the boundaries of the signal peptide, of the SU (surface unit) subunit and of the TM (Trans membrane) subunit and the receptor binding site. The sequences are HERV-W (Human Endogenous Retroviral Family W), RD114 (Cat Endogenous retrovirus), REV (Avian Reticuloendotheliosls Virus), BAEV (Baboon endogenous virus (strain M7)), SRVl (Simian retrovirus SRV-1), SRV2 (Simian retrovirus SRV-2) and MPMV (Simian Mason-Pfizer virus).
The following examples are given by way of illustration and have no limiting character. They will make it possible to better understand the invention.
Example 1: Molecular and phenotypical characterization of recombinant envelopes
Construction and production of the HERV-W envelope SU subunit
A vector phCMVEnv-Gp60 allowing the expression of a soluble recombinant envelope protein was designed from the expression vector phCMV-Env-W (Blond J Virol, Vol 74(7): 3321-3329, 2000) containing the HERV-W envelope gene (538 amino acids) (clone PH74, Blond et al. J Virol Vol 73(2): 1175-1185, 1999).
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The soluble envelope (Gp60,1-435) was constructed as described below:
(1) The native cleavage site RNKR (AA 314 to 317)
between the SU and TM subunits was mutated to AAAR in
order to allow the production of a fusion protein that
was stable and not of two SU-TM subunits cleaved and
then recombined by a disulfide bridge.
(2) The transmembrane (tm) and intracytoplasmic
(CYT) regions corresponding to amino acids 436 to 538
were deleted in order to obtain a soluble protein.
(3) A spacer arm having the composition (GGGS)3,
followed by a polyhistidine tail (RGS-HHHHHH), were
added at the C-terminal position in order to allow the
purification of this protein by IMAC and the detection
by an anti-histidine monoclonal antibody (Qiagen,
RGS H6) .
Starting with the vector phCMVEnv-Gp60 expressing the soluble envelope, the vector phCMV-EnvSU was constructed, allowing the production of an SU protein. The soluble SU is a fusion protein containing a C-terminal polyhistidine tail having the sequence RGS-HHHHHH immediately downstream of the sequence AAAR, in order to allow the purification of this protein by IMAC and the detection by an anti-histidine monoclonal antibody (Qiagen, RGS H6) .
The schematic structure of the various proteins produced from the vectors phCMV-Env-W, phCMV-EnvGp60 and phCMV-EnvSU is illustrated in figure la.
Production of the soluble envelope
The expression plasmid phCMV-EnvGp60 or phCMV-EnvSU is transfected into the HEK293T cells by precipitation with calcium phosphate. The supernatant containing the GP60 or SU envelope is collected after 48 hours of
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production in a serum-free medium and filtered on 0.45 µm membranes in order to remove the cellular debris. 20 µ1 of supernatant are directly analyzed on a polyacrylamide gel and by Western blotting with an anti-histidine monoclonal antibody (Quiagen, RGS H6) . The GP60 and SU proteins are correctly expressed in soluble form.
Binding test and analysis by flow cytometry
The stable lines XChASCT2 and XChASCTl constitutively expressing the hASCT2 (XChASCT2) or hASCTl (XChASCTl) receptors were established after transfection of XC cells (rat sarcoma) with vectors expressing either human receptor hASCT followed by selection of a clone as described above (Frendo et al. , Mol. Cell Biol,, Vol 23(10): 3566-3574, 2003). The following human cells are described in Blond J Virol, Vol 74(7): 3321-3329, 2000. The TE671 cells express hASCT2. The TE671RD cells constitutively express the RD114 envelope (cat endogenous retrovirus) belonging to the same interference group and therefore recognizing the hASCT2 receptor. TE671galv cells constitutively express the GALV (gibbon ape leukemia virus) envelope belonging to another interference group and recognizing the PiT1 receptor.
The cells were washed in PBS and harvested by detaching with 0.02% versene in PBS. A total of 10 cells were incubated with 1 ml of filtered supernatant containing the soluble envelope (Gp60 or SU) for 1 hour at 37°C. The cells were washed with PBA (PBS and 0.5% sodium azide) containing 2% fetal calf serum and were labeled for 1 hour at 4°C with an anti-histidine monoclonal antibody (RGSH6, Quiagen). The cells were washed once with PBA and incubated with a secondary antibody coupled to fluorescein isocyanate for 1 hour at 4°C. The cells were washed twice with PBA and analyzed by
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flow cytometry.
Using the target cells XChASCT2, the inventors demonstrated that the recombinant protein Gp60 corresponding to a soluble form of the envelope has a phenotypical characteristic identical to that of the wild-type envelope, namely that it is capable of binding to XC cells expressing the hASCT2 receptor.
Using the target cells XChASCT2, XChASCTl, TE671, TE671RD, TE671 galv, the inventors demonstrated that the recombinant protein corresponding to the SU subunit of the envelope exhibits phenotypical characteristics identical to those of the wild-type envelope. First of all, the SU subunit is capable of binding to two receptors hASCTl and hASCT2 (figure lb). Furthermore, this protein was tested in relation to human cells TE671 and derived cells TE671RD and TE671galv. The soluble SU protein bound to the TE6781 cells expressing the hASCT2 receptor and the TE671galv cells blocked for the PiTl receptor, but did not bind to the TE671RD cells blocked for the hASCT2 receptor (figure Ic) . The SU recombinant protein and the envelope of the RD114 retrovirus specifically interfered.
Example 2: Identification of the domains for interaction of the SU part of the W envelope with its hASCT2 receptor
In order to identify the boundaries of the region of the envelope binding to the hASCT2 receptor, the inventors constructed a set of deletion mutants from the N- and C-terminal ends. The domains of the SU subunit were obtained by PCR and subcloned into the expression vector pHCMV-EnvSU and sequenced. The expression plasmids phCMV-EnvSU, Env69-317, Envl97, Envl68, Envl69-317, Envll7 and Envl44 (figure 2a) were transfected into the HEK293T cells by precipitation
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with calcium phosphate. The conditions for production and analysis of the proteins are identical to those detailed in example 1.
EnvSU, Env69-317 and Envl97 proteins were correctly
expressed in soluble form. Using the XC cells
constitutively expressing hASCT2, the inventors
emonstrated that the Envl97 protein was capable of
ending to the receptor expressed at the surface of the
clls like the SU subunit (1-317). Thus, the first 176
esidues of the mature SU subunit (and therefore
acking its signal peptide) were sufficient for binding
a the surface of the cells expressing the hASCT2
eceptor. The deletion of the 21-68 region resulted in
loss of binding to the receptor also indicating its
evolvement in the receptor binding domain (RBD) . On
he other hand, the truncated Envl68 protein showed a
power capacity for binding to the hASCT2 receptor.
order to obtain the equivalent quantities in the upernatants between the various truncated proteins, the inventors fused two smaller domains of the -terminal region of SU (Envll7 and Envl44) to the -terminal region of the SU subunit (Envl69-317), the atter domain not binding to hASCT2. The level of kpression of the Envll7 and Envl44 proteins was Lmilar and the proteins were expressed in soluble form. The binding test showed that only the Envl44 rotein was capable of binding to the cells expressing he hASCT2 receptor. The absence of binding of the nvll7 protein to the surface of the cells indicated he loss of at least one determinant of binding inside the 117-144 region.
pnsequently, the boundaries of the domains for proteraction of the W envelope with its receptor are efined by amino acids 21 to 144.
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It should be noted that, in general, proteins (including the envelope proteins) intended for secretion or for membrane expression are synthesized in the granular endoplasmic reticulum (ER) . The translocation of the neosynthesized proteins in the ER is conditioned by an N-terminal signal peptide (Walter and Lingappa 1986). The hydrophobic region of the signal peptide initiates penetration into the membrane of the reticulum, bringing behind it the remainder of the neosynthesized peptide. Since the translocation starts at the same time as the synthesis, it is the peptide being translated which crosses the ER membrane. While the protein passes into the lumene of the ER, the signal sequence is cleaved by a specific cellular enzyme, signal peptidase (Walter P, Johnson AE: Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 1994, 10: 87-119). The translocation of Env into the ER is stopped by the (hydrophobic) transmembrane domain of the glycoprotein which becomes anchored at the phospholipid membranes. In the ER lumene, the regions (SU and part of TM) intended to become extracytoplasmic are folded (the disulfide bridges formed), glycosylated and oligomerized. After oligomerization, the proteins undergoing maturation are transported into the Golgi apparatus where they undergo new glycan maturation processes and cleavage by furin-type endoproteases recognizing a motif R/KXXR leading to two SU and TM subunits.
The mature protein is targeted to the plasma membrane by virtue of a motif present on the intracytoplasmic tail containing a tyrosine (aliphatic/aromatic(Y-X-X)).
Example 3: Test of In vitro inhibition of the binding of the envelope to its receptor (definition of a peptide and generation of a rabbit antibody)
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A peptide (112-129, TGMSDGGGVQDQAREKHV+C, 19 amino acids) was defined from the region defined in example 2 and from a determination of the potentially antigenic regions of the SU subunit. A cysteine was added at the C-terminal position for the KLH (keyhole limpet hemocyanin, cf. Frendo et al., Mol. Cell Biol. Vol 23(10): 3566-3574, 2003) coupling. This peptide was used to immunize a rabbit and then to affinity purify the polyclonal antibody directed against the region 112-119 contained in the serum of this rabbit.
The Envl44 protein is preincubated at 37°C for one hour with either the anti-SU polyclonal antibody or with an anti-TM polyclonal antibody. The formation of the Envl44 protein-anti-SU antibody complex drastically reduced the binding of the envelope to the cells expressing the hASCT2 receptor. By contrast, the use of an antibody not directed against the RBD did not adversely affect its binding to the hASCT2 receptor.
Example 4: Production of monoclonal antibodies directed against the HERV-W envelope protein
Immunization of mice with DNA
Three female six-week-old BALB/c mice (IFFA-Credo) were immunized by direct injection of naked plasmid DNA (phCMV-env-W) containing the gene for the HERV-W envelope. The injections were performed by the
intradermal route with the aid of a gene gun. Five injections of 2 µg of DNA were first performed for each mouse followed by a booster with two injections of 4 µg of DNA. The sera were collected and the antibody titer for each serum was determined. Since the antibody titer
was too low, a cellular lysate was prepared.
Preparation of the cellular lysate
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The rhabdomyosarcoma cells TelCeB6 (ATCC CRL8805) were transfected with the plasmid phCMV-env-W. After about 20 hours and the presence of syncytia, a cellular extract was prepared in PBS buffer containing 0.5% Triton. The protein extracts were assayed by Bradford. The env-W antigen concentration corresponded to 9.5 pg/yl of total proteins.
Immunization of mice with an extract of cellular lysate
The same mice first of all received an injection of 10 ]jg of cellular lysate by the intraperitoneal route followed by a booster injection of 2 x 100 µg of cellular lysate by the intraperitoneal route. Three days before the fusion with myeloma cells, another injection was performed,-by the intravenous route, with 22 µg of soluble envelope protein Gp60 obtained from the plasmid phCMV-Env-Gp60 as described in example 1, purified beforehand, before injection, on to an Ni-NTA resin (Quiagen) according to the following conditions: binding in phosphate buffer pH 8, washes in phosphate buffer pH 8 and in ammonium acetate pH 6, elution in ammonium acetate buffer pH 3.5 and concentration with speed vac. 47 jjg of the eukaryotic Gp60 protein thus obtained were reserved for the injection by the intravenous route described above. After fusion, the hybridoma supernatants were tested by immuno¬fluorescence on the transfected and bound cells (TeLCeb6), the antibodies were screened by a functional ELISA test using the Env-W protein at a concentration of 9.2 µg/µl of total proteins and an Env AS protein as negative control at a concentration of 13.3 µg/µl of total proteins and the most effective antibodies were selected.
The monoclonal antibodies 2H1H8, '12C7A3 and IF11B10 were thus obtained. The monoclonal antibodies 2H1H8 and 12C7A3 are directed against the nonglycosylated N-
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terminal part of the SU region of the Env-HERV-W proteins. They are directed against the RDB as shown by a Western blot assay with the aid of Env 144. The monoclonal antibody IF11B10 is directed against the glycosylated C-terminal part of the SU region of the Env-HERV-W protein as shown by a Western blot assay with the aid of Env 169-317. It does not recognize Env 144.
Example 5: Test of in vitro inhibition of the binding of the envelope to its receptor and inhibition of the formation of syncytia with the aid of monoclonal antibodies by a cell to cell fusion test (coculture)
The plasmid for expressing the envelope glycoprotein is transfected into the cells TELCeB6 by precipitation with calcium phosphate at two quantities 100 and 500 ng (Cosset-et al., Journal of Virology, 69 (10): 6314-6322 (1995)). The cells expressing the envelope are detached from the support 20 hours after transfection and are preincubated at 37 °C for one hour, respectively, with the anti-HIV 23A5 monoclonal antibody, the anti-TM Env-HERV-W 6A2B2 monoclonal antibody (previously obtained), the anti-SU Env-HERV-W 2H1H8 12C7A3 and IF11B10 monoclonal antibodies (dilution l/50th). Next, they are reinoculated at equal concentration (0.4 x 105 cells/well) into 12-well plates. Epithelioid-carcinoma-indicating human cells (Hela, ATCC CCL-2) are then added to the transfected cells in an amount of 2 X 10^ cells per well and the co-culture is continued for 24 h. An XGal (5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside) staining may then be performed in order to stain the nucleus of the cells TELCeB6 (Cosset et al., Journal of Virology, 69 (10): 6314-6322 (1995) ) . It is followed by staining with May-Griinwald and Giemsa solutions (MERCK) performed according to the suppliers' recommendations. The fusion observed corresponds to a fusion "from within", that is to say a
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cell to cell fusion, starting with a cell expressing the envelope, in contrast to a fusion "from without" which corresponds to a formation of syncytia following a virion-cell (s) fusion.
The results presented in table 1 below express the number of fused cells counted.

The results presented above show that the formation of the Env protein-anti-SU 2H1H8 and 12C7A3 antibody complex dramatically reduces the binding of the envelope to cells expressing the hASCT2 receptor and cell fusion. By contrast, the use of an antibody not directed against rdb (6A2B2 or IFIIB10) does not adversely affect the binding of the envelope and the fusion of the cells significantly, as may be observed by comparing to the results obtained with the anti-HIV 23A5 control antibody.
Example 6: Study of the in vivo interaction between the envelope protein and the hASCT2 receptor and the in vivo formation of syncytia
To verify the results obtained in vitro, an animal model was designed. Rhabdomyosarcoma cells TelCeB6 (ATCC CRL8 805), in culture in DMEM medium (Gibco Invitrogen 41966-029) supplemented with South American serum, were respectively transfected, with the aid of the LipofectAMINE PLUS™ kit (Gibco Invitrogen), with the DNA corresponding to the HERV-W env gene cloned in the sense orientation at the concentration of 2 µg/µl
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(DNA 409), with the DNA corresponding to the HERV-W env gene cloned in the anti-sense orientation at the concentration of 1.5 µg/µl (DNA 410) and with the DNA corresponding to a mutated HERV-W env gene at the concentration of 1.3 µg/µl (DNA LQMV) according to the protocol detailed below. The cells transfected with the DNA 409 are capable of expressing the fusogenic W envelope protein, the cells transfected with the DNA 410 are not capable of expressing the envelope protein and the cells transfected with the DNA LQMV are capable of expressing a nonfusogenic mutated W envelope protein.
1). Protocol:
1st Day: Culture of the TelCeB6 cells:
- inoculation of the dishes 100 mm in diameter —> 50-
70% confluence;
- incubation in supplemented DMEM medium (6 ml per dish) for 24 hours at 37°C under 5% CO2.
2nd Day: Transfection with the LipofectAMINE PLUSTM kit:
1 Precomplexing of the DNA
- mixing of 750 µ1 of medium not supplemented with the
abovementioned DNAs in a 15 ml falcon tube (reference
2096), that is 2 µ1 of 409 or 3 µ1 of 410 or 3 µl LQMV;
- stirring under vortex of PLUS reagent and adding 20 µ1 thereof to the DNA solution;
- stirring under vortex immediately 10 seconds at
1400 rpm;
- incubation 15 minutes at room temperature.
5
2 Preparation of the cells
- replacement with 5 ml of nonsupplemented medium.
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3 Dilution of the LipofectAMINE
In a tube for a dish, mix 30 µ1 of LipofectAMINE Reagent with 750 µ1 of nonsupplemented medium.
4 Complexing of the DMA
Mixing of 780 µ1 of dilute LipofectAMINE and 772 µ1 of solution of precomplexed DNA (total: 1552 µ1) ; stirring under vortex immediately 10 sec at 1400 rpm, incubation 15 minutes at room temperature.
5 Transfection and production of recipient animals
transplanted with the target cells, treated or not
treated by injection of anti-Env antibody
Deposition of 1552 µ1 in a dish;
incubation 2-3 hours at 37°C under 5% CO2;
replacement of the transfection medium with 6 ml of
supplemented medium;
incubation 1 hour at 37°C under 5% CO2;
injection by the intraperitoneal route (IP) to SCID
mice (in a volume of 1 ml) , l/5th of each dish at 70%
confluence, with or without additional injection of
anti-Env protein antibody (monoclonal antibody 2H1H8,
polyclonal antibody 69 (anti-SU) and 71 (anti-TM) at
1/100).
Production of animals tolerating the transplant and
allowing dissemination of the transplanted cells in the
body, in parallel with the establishment of pseudo-
ascites in the peritoneal cavity.
3rd Day:
Collection of the cells from each animal by peritoneal lavage: injection of 2 ml of air followed by 2 ml of physiological saline and then massaging and recovering
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the 2 ml of peritoneal fluid (protocol developed for the recovery, in transplanted animals, of the cells implanted in the peritoneal cavity);
observation under an "inverted phase" microscope with counting of the syncytia and/or after staining on a slide;
immediate reading performed after spreading on slides with a gridded chamber in the presence of Trypan blue (exclusion of the dead cells) . The number of cells which have fused to each other was counted per field with a "wide angle" lens (40) which makes it possible ... to establish the count on more than about one hundred cells so as to have a series of statistically representative counts.
A cellular aliquot of each sample is fixed in the presence of methanol/acetone (v/v) and then stored at -20°C until a crystal violet staining is obtained (1%). Photographs were taken of the stained slides.
2) Mice:
Groups of 2 mice are inoculated with:
the cells transfected with the three types of plasmid
(DNAs 409, 410 and LQMV) with no antibody (3x2= 6 mice) the cells transfected with the three types of plasmid
(DNAs 409, 410 and LQMV) with the monoclonal antibody 2H1H8 (3x2= 6 mice).
3) Results:
The number of fused cells determined by direct reading on a gridded counting chamber per 100 cells is indicated in Table 2 below:
Table 2
ECP (Tryptan blue) reading: direct reading of the REPLACEMENT SHEET (RULE 26)

Each number represents the number of fused and visualized cells per field studied. As some cells may be superposed in the optical path, the count for the cells appearing fused in the controls is greater than zero. The reality of the syncytia and the discrimination with stacks of cells were then verified by staining the cells on a slide, with visualization of multiple cell nuclei contained in a space delimited by the continuation of a single and sole cell membrane. Moreover, photos showing cells in the course of fusion made it possible to objectify the reality of the fusion upon analysis by phase contrast microscopy and the total absence of an equivalent phenomenon in the controls.
To statistically objectify the primary analysis represented by the numbers indicated in Table 2, a Chi-2 test was performed in order to compare the data in Table 2.
The results of the statistical analysis taking into account the "background noise" of the primary reading, without secondary analysis after staining on a slide or a search for typical cells in the course of fusion which are never seen in the controls, are the following:
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i) Statistical validation of the specificity of the
pathogenic effect in vivo:
Env expressed (409): 20.5 positives counted on average
out of 100 cells,
anti-sense Env (410): 9.5 positives counted on average
out of 100 cells,
mutated Env (LQMV): 6.5 positives counted on average
out of 100 cells,
mean of the controls (410 and LQMV): 9.5 + 6.5/2 = 8%.
Env versus control 410: Chi-2 = 5.89 (p Env versus control LQMV: Chi-2 =9.83 (p Env versus the two controls (410 and LQMV): Chi-2 = 7.69
(p Control 410 versus control. LQMV: Chi-2 =0.61
(difference not significant).
The controls are therefore statistically equivalent and there is no "real" difference linked to the type of control.
The results obtained from this stage of analysis (not excluding the background noise linked to the artefactual images and by comparing the two types of control with each other (which prove to be equivalent)) was statistically very significant (overall p ii) Statistical validation of the therapeutic activity of the antibodies tested on the pathogenic effect in vivo:
Env expressed (409): 20.5 positives counted on average out of 100 cells,
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Env expressed (409) + monoclonal antibody 2H1H8: 3.5 positives counted on average out of 100 cells.
Env alone versus injection of the antibody 2H1H8: Chi-2 = 15.38 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of result due to chance (p) less than 0.001). Subsequent analyses, by staining, of the specificity of the effects merely confirm the specificity of the effect obtained in vivo in the presence of the Env protein and of antibody, thereby validating the therapeutic effect on the animal model.
Example 7: Study in vivo of the binding of the HERV-W Env protein to cells possessing or not possessing type 1 or 2 hASCT receptors and of the inhibition of this binding by injection of antibodies directed against HERV-W Env
1) Materials
Soluble protein: supernatant filtered on 0.4 5 µm
containing the soluble protein (293T cells transfected
with the. plasmid 460 (envelope-spacer-His6) .
Expression verified by Western blotting with an anti-
RGS-His antibody.
Antibody: monoclonal antibody 2H1H8 (IgG, 5.50 mg/ml). Cells: XChASCT2, cellular clone XC (ATCC CCL-165, rat
cells) expressing the hASCT2 receptor.
DMEM medium (Gibco Invitrogen 41966-029) with South
American serum.
Preincubation, incubation, labeling in a 1.5 ml Eppendorf tube.
2) Protocol
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1 IP (intraperitoneal) inoculation of the XChASCTl,
XChASCT2 cells and control cells XChASCT - into SCID
mice
Injection into mice of l/5th of the flask at 70% confluence in a volume of 2 ml.
2 Preincubation
Incubation of the soluble protein supernatant (filtered supernatant of the 293T line) with the monoclonal antibody 2H1H8 (990 µ1 of supernatant with 10 µ1 of antibody (1/lOOth dilution)) for 1 hour at 37°C in the cell incubator with occasional stirring (every 15 minutes).
3 Inoculation
IP (intraperitoneal) inoculation of the proteins alone or with the antibody into mice transplanted with the cells (1 X 106 cells per point, that is l/5th of a confluent dish 100 mm in diameter).
After injection of the antibody (200 microliters), maintained as IP, for 6 hours, with an occasional peritoneal massage (every 30-60 minutes).
4 Recovery of the cells by peritoneal lavage of the
transplanted mice
- Centrifugation 3000 revolutions for 5 minutes at + 4°C.
- Recovery of the cellular pellet and dilution in the labeling media (maintained at +4°C until fixing).
5 Labeling
- Primary antibody:
The pellet is taken up in 100 µ1 of anti-RGS His antibody (100th dilution - Quiagen) in a PBA buffer (PBS with 2% fetal calf serum and 0.1% sodium azide) , maintained at +4°C.
1 hour in ice with occasional stirring (every 15 minutes) .
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Washing in PBA buffer (1 ml per tube), maintained at + 4°C.
- Secondary antibody:
Centrifugation 3000 revolutions for 5 minutes at +4°C. The pellet is taken up in 100 µl of anti-mouse antibody-FITC (l/20th dilution - DAKO, reference F0479 in a PBA buffer) maintained at +4°C.
1 hour in ice with occasional stirring (every
15 minutes).
2 washes in PBA buffer (1 ml per tube), maintained at
+ 4°C.
Pellet taken up in 500 ]il of PBA, maintained at'+4°C, and analyzed by FACS.
Alternatively, analysis by IF after fixing on a slide in acetone/methanol (50%/50%) at -20°C and counter-staining with Evans blue.
3) Mice:
Groups of 2 mice are inoculated with:
each type of cell (expressing the 2 types of receptor hASCTl and hASCT2 and one not expressing the receptor hASCT - as a control) with no antibody (3x2 = 6 mice) the three types of cell with the Env protein and the monoclonal antibody 2H1H8 (3x2 = 6 mice).
4) Results
The results by immunofluorescence (IF) reading with a microscope are presented in Table 3 below:

Each number represents the number of cells visualized as fluorescent per field studied. As some cells may have bound fluorescence in a non-specific manner, the count for the cells appearing fluorescent under the control conditions is therefore greater than zero in one of the two fields counted (mean of the two fields = 1/28, that is 0.036%, which is entirely reasonable for the background noise of such a reading technique). The reality of the cells that bound the Env protein to their hASCTl or hASCT2 receptor was then verified by cytofluorometric analysis.
In order to statistically objectify the analysis presented in Table 3, a Chi-2 test was performed in order to compare the data obtained under the conditions according to which (i) the Env protein can bind to a receptor hASCTl (control hASCTl) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the grafted control cells which have no receptor (control X) to which the Env protein injected into the corresponding animals cannot bind and thus does not give membrane fluorescence in the presence of an anti-Env antibody and (ii) the Env protein can bind to a receptor hASCTl (control hASCTl) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the injection of a monoclonal antibody directed against Env-SU.
The results of the statistical analysis are presented
below:
i) Statistical validation of the specificity of the
pathogenic effect in vivo:
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control hASCTl: 24 positives counted on average out of
77 cells,
control hASCT2: 23 positives counted on average out of
57 cells,
hASCT- cells: 1 positive counted on average out of 28
cells.
Env+grafts hASCTl versus hASCT-: Chi-2 = 8.62 (p The cells expressing the hASCTl or hASCT2 receptors at their surface are therefore indeed statistically equivalent and there is no difference in the Env binding to the receptor linked to subtype 1 or 2, under the conditions of the experiment.
The results obtained with the animals transplanted with the cells expressing the membrane receptors hASCTl or hASCT2 are statistically significant in the light of the results obtained with the control animals trans¬planted with the cells expressing none of these receptors at their surface.
These results confirm the specificity of the effect obtained in vivo in the presence of the Env protein in the animal models.
ii) Statistical validation of the therapeutic activity
of the antibodies tested on the pathogenic effect
in vivo:
Env+grafts control hASCTl: 24 positives counted on
average out of 77 cells
Env+grafts hASCTl + monoclonal antibody 2H1H8: 2
positives counted on average out of 73 cells.
Env+grafts hASCTl alone versus injection antibody REPLACEMENT SHEET (RULE 26)

2H1H8: Chi-2 = 21.14 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) less than 0.001).
Env+grafts hASCT2: 23 positives counted on average out of 57 cells
Env+grafts hASCT2 + monoclonal antibody 2H1H8: 1 positive counted on average out of 45 cells.
Env+grafts hASCT2 alone versus injection antibody 2H1H8: Chi-2 = 20.31 (p The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) overall less than 0.01).
The validation of the animal models against the appropriate controls makes it possible to demonstrate that antibodies may have a therapeutic activity by significantly inhibiting the pathogenic effects of the ~HERV-W Env protein.
Example 8: Alignment of the sequences of the interference group
The protein sequences of the envelopes of the retroviruses HERV-W (swiss-prot Q9UQF0), RD114 (swiss-prot Q98654), REV (swiss-prot P31796), BAEV (swiss-prot P10269), SRVl (swiss-prot P04027), SRV2 (swiss-prot P51515) and MPMV (swiss-prot P07575) were aligned with the aid of the MacVector software with the ClustalW procedure. The signal peptide, the SU (surface unit) subunit and the TM (Trans membrane) subunit are indicated. The receptor binding site is underlined.
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CLAIMS
1. A peptide domain necessary for the interaction between the envelope of a virus belonging to the HERV-W pterference group and an hASCT receptor, defined in hat it starts with an N-terminus and ends with a -terminus, and in that:
he N-terminus is defined by a motif chosen from SEQ ID 1 to SEQ ID No. 29,
he C-terminus is defined by a motif chosen from SEQ No. 30 to SEQ ID No. 40,
and in that said peptide domain comprises, between the -terminus and the C-terminus, at least one motif hosen from SEQ ID No. 41, SEQ ID No. 42 and SEQ ID . 73.
A nucleotide sequence encoding a peptide domain as fined in claim 1.
An epitope derived from the peptide domain as ifined in claim 1, characterized in that it induces an amune response against a virus belonging to the HERV-W iterference group.
A nucleotide sequence encoding an epitope as ifined in claim 3.
An expression vector, characterized in that it emprises a nucleotide sequence as claimed in either of aims 2 and 4, and the means necessary for its pression.
A microorganism or host cell transformed with at east one expression vector as defined in claim 5.
An antibody directed against a peptide domain as

defined in claim 1 or against an epitope as defined in claim 3.
8. The use of at least one peptide domain as defined in claim 1 or of at least one epitope as defined in claim 3, or of at least one antibody as defined in claim 7 or of at least one nucleotide sequence as defined in claims 2 or 4 placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, for the preparation of a medicament intended for the inhibition, prevention or treatment of an infection caused by a virus belonging to the HERV-W interference group in an animal, preferably humans.
9. A pharmaceutical composition comprising, by way of active substance, at least one peptide domain as defined in claim 1, or at least one epitope as defined in claim 3, or alternatively at least one of the nucleotide sequences as defined in claims 2 or 4, placed under the control of elements necessary for a constitutive and/or inducible expression of said peptide domains or epitopes, or alternatively at least one antibody as defined in claim 7 in combination with a pharmaceutically appropriate vehicle.
10. A diagnostic composition for the detection and/or quantification of a virus belonging to the HERV-W interference group and/or the quantification of an immune response against a virus belonging to the HERV-W interference group, comprising at least one peptide domain as defined in claim 1, at least one epitope as defined in claim 3, or at least one of the nucleotide sequences as defined in claims 2 or 4, or at least one antibody as defined in claim 7.
11. A method for the detection and/or quantification of a virus belonging to the HERV-W interference group
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in a biological sample taken from an individual liable to be infected by said virus, characterized in that it comprises the steps consisting in:
bringing said biological sample into contact with at least one antibody as defined in claim 7 under conditions allowing the formation of a complex between the virus and the antibody, and
detecting and/or quantifying the formation of said complex by any appropriate means.
12. The use of the composition as claimed in claim 10 for the in vitro screening of a virus belonging to the HERV-W interference group in a biological sample or specimen.
13. The use of a peptide domain as claimed in claim 1 or of an epitope as defined in claim 3, or of an antibody as defined in claim 7, for inhibiting the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor.
14. The use of a peptide domain as claimed in claim 1, for identifying chemical or biological molecules whose interaction with all or part of this peptide domain blocks the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor.
15. The use of a peptide domain as claimed in claim 1 for generating antibodies blocking the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor.
16. A method for determining a polypeptide region necessary for the interaction between the envelope of a virus belonging to the HERV-W interference group and an hASCT receptor, characterized in that:
the nucleotide and/or peptide sequence of the
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precursor envelope of said virus is identified
the signal part is excluded
a serine-aspartic acid-Xa-Xb-Xc-Xd-Xg-aspartic
acid-Xf-Xg motif is detected, which corresponds
to SEQ ID No. 4 3
in which Xa, Xb, Xc, Xd, Xg, Xf, Xg are any amino acids
the C-terminus is excluded between 15 and 25
amino acids after said serine-aspartic acid-Xg-
Xb-Xc-Xd-Xg-aspartic acid-Xf-Xg motif, which
corresponds to SEQ ID No. 43.
17. A peptide domain which can be obtained by the method as claimed in claim 16.

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Patent Number

272906

Indian Patent Application Number

4129/CHENP/2008

PG Journal Number

19/2016

Publication Date

06-May-2016

Grant Date

02-May-2016

Date of Filing

06-Aug-2008

Name of Patentee

BIO MERIEUX

Applicant Address

CHEMIN DE I ORME, 69280 MARCY-L- ETOILE,

Inventors:

#	Inventor's Name	Inventor's Address
1	MALLET, FRANCOIS	84 RUE ANATOLE FRANCE, 69100 VILLEURBANNE,
2	ORIOL, GUY,	13 RUE JEAN- BAPTISTE RIVORY, 42400 SAINT CHAMOND,
3	CHEYNET, VALERIE,	LES AULLIEUX, 42410 VERIN,

PCT International Classification Number

C07K14/15

PCT International Application Number

PCT/FR07/00236

PCT International Filing date

2007-02-09

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0650468	2006-02-09	France