US20090148454A1

US20090148454A1 - Peptide Domain Required For Interaction Between The Envelope of a Virus Pertaining to The Herv-W Interference Group and an Hasct Receptor

Info

Publication number: US20090148454A1
Application number: US12/087,893
Authority: US
Inventors: Francois Mallet; Guy Oriol; Valerie Cheynet
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2006-02-09
Filing date: 2007-02-09
Publication date: 2009-06-11
Also published as: CA2640793C; DK1981904T3; AU2007213591A1; IL193353A0; EP1981904B1; JP6494487B2; ES2637961T3; CN101379079B; US10040829B2; CA2640793A1; JP2016040295A; US20160090402A1; EP1981904B9; AU2007213591B2; JP2009525741A; WO2007090967A2; NO20083549L; HUE034297T2; CN101379079A; EP1981904A2

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 a 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)-proline-cysteine-X-cysteine in which Z is any amino acid, 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) in which Xa, Xb, Xc, Xd, Xe, Xf, Xg are any amino acids, Z is any amino acid, B 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 pattern selected from the following patterns: a pattern formed by the amino acids cysteine-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.

Description

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 ERVW1 and synthesized in the form of a gPr73 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, hASCT1). 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 hASCT2 (Rasko et 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 L P., T-Ag inhibits implantation by EC cell derived embryoid bodies. Virus Genes. 2000; 20(3): 195-200; J E, Battini J L, Gottschalk R J, Mazo I, Miller A D., 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 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)_α-proline-cysteine-X-cysteine in which
  - Z is any amino acid
  - α 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 the amino acids serine-aspartic acid-X_a-X_b-X_c-X_d-X_easpartic acid-X_f-X_g-(Z)_pin which
  - X_a, X_b, X_c, X_d, X_e, X_f, X_gare any amino acids
  - Z is any amino acid
  - β 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:
- a motif consisting of the amino acids cysteine-tyrosine-X₂-X₃-X₄-X₅-X₆-cysteine, in which X₂, X₃, X₄, X₅, X₆are 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, X₁₅are 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 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, α 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, X_a, X_b, X_cor the amino acids at positions 3, 4 and 5 of SEQ ID Nos. 30 to 40 are a glycine, X_dor the amino acid Xaa at position 6 of SEQ ID Nos. 30 to 40 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; X_for 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, X_gor the amino acid at position 10 of SEQ ID Nos. 30 to 40 is an amino acid chosen from alanine, lysine, isoleucine, threonine and valine.
Preferably, β is an integer equal to 20.
Preferably, X₂or the amino acid Xaa at position 3 of SEQ ID No. 41 is an amino acid chosen from asparagine, threonine, glutamic acid, histidine, X₃or the amino acid Xaa at position 4 of SEQ ID No. 41 is an amino acid chosen from histidine, alanine, serine, lysine, glutamic acid; X₄or the amino acid Xaa at position 5 of SEQ ID No. 41 is an amino acid chosen from tyrosine, threonine, alanine, X₅or the amino acid Xaa at position 6 of SEQ ID No. 41 is an amino acid chosen from glutamine, arginine, threonine, X₆or the amino acid Xaa at position 7 of SEQ ID No. 41 is an amino acid chosen from leucine, glutamine, glutamic acid.
Preferably X₇or the amino acid Xaa at position 2 of SEQ ID No. 42 is an amino acid chosen from proline, threonine, arginine and asparagine; X₈or the amino acid Xaa at position 3 of SEQ ID No. 42 is an amino acid chosen from glycine, glutamic acid, asparagine, Xg or the amino acid Xaa at position 4 of SEQ ID No. 42 is an amino acid chosen from glycine, asparagine, isoleucine, threonine, serine, X₁₀or the amino acid Xaa at position 5 of SEQ ID No. 42 is lysine or is deleted; X₁₁or the amino acid xaa at position 6 of SEQ ID No. 42 is an amino acid chosen from lysine, valine, isoleucine, leucine, X₁₂or the amino acid Xaa at position 7 of SEQ ID No. 42 is an amino acid chosen from glycine, asparagine; X₁₃or the amino acid Xaa at position 8 of SEQ ID No. 42 is an amino acid chosen from glutamine, lysine, valine, X₁₄or the amino acid Xaa at position 9 of SEQ ID No. 42 is an amino acid chosen from valine, proline, serine, threonine, X₁₅or the amino acid Xaa at position 10 of SEQ ID No. 42 is an amino acid chosen from valine, isoleucine.
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, X₁₀is 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, 1989.
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 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.
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, Schwani omyces, Zygosaccharomyces; Saccharomyces cerevisiae, Saccharomyces carlsbergensis and Kluveromyces lactis being preferred; and bacteria such as E. coli and those of the following families: Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus and 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 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.
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 R R, 1959, Biochem. J., 73: 199-126) or of the F(ab)′₂type (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.
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 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 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.
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 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 a patient 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 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 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, β-galactosidase, glucose-6-phosphate dehydrogenase,
- chromophores such as luminescent, coloring compounds,
- radioactive molecules such as 32P, 35S or 125I,
- fluorescent molecules such as fluorescein, rhodomine, alexa or phycocyanins, and
- particles such as gold or magnetic latex particles, liposomes.

Indirect labeling systems may also be used, such as, for example, via another ligand/anti-ligand pair. The ligand/anti-ligand pairs are well known to a person skilled in the art, and the following pairs may be mentioned for example: biotin/streptavidin, biotin/avidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/complementary strand for the polynucleotide. In this case, it is the ligand which is bound to the binding partner. The anti-ligand may be detected directly by the markers described in the proceeding paragraph or may itself be detected by a ligand/anti-ligand.
These indirect systems may lead, under certain conditions, to an amplification of the signal. This signal amplification technique is well known to a person skilled in the art, and reference may be made to the previous patent applications FR98/10084 or 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, Calif. 92101 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, N.Y.
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 SRV1 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 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 molecules 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 molecule alters the env/receptor interaction. Alternatively, 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 intracytoplasmic 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:

- the nucleotide and/or peptide sequence of the precursor envelope of said virus is identified
- the signal part is excluded
- a serine-aspartic acid-X_a-X_b-X_c-X_d-X_e-aspartic acid-X_f-X_gis detected domain in which
- X_a, X_b, X_c, X_d, X_e, X_f, X_g, 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-X_a-X_b-X_c-X_d-X_e-aspartic acid-X_f-X_gdomain, which corresponds to SEQ ID No. 43.

Preferably, X_a, X_b, X_cis an amino acid which is glycine, X_dis an amino acid chosen from proline and valine, X_eis an amino acid chosen from glutamine, leucine and threonine, X_fis an amino acid chosen from lysine, threonine, methionine and glutamine, X_gis 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 Dyrløv Bendtsen, Henrik Nielsen, Gunnar von Hiejne and Søren 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 S F, Gish W, Miller W, Myers E W, Lipman D J., 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.

FIG. 1 illustrates the phenotypical characteristics and properties of soluble recombinant proteins derived from Env-W. In particular, FIG. 1 a 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). FIG. 1 b represents the flow cytometry analysis of the test for binding of the EnvSU recombinant protein to XC hASCT2 and XC hASCT1 cells expressing the hASCT2 and hASCT1 receptors, respectively. FIG. 1 c illustrates the test of interference of binding to the TE671 cells (control hASCT2), TE671RD cells (blocked hASCT2) and TE671galv cells (blocked Pit1).

FIG. 2 illustrates the definition of the minimum binding domain of the ERV-W envelope to the hASCT2 receptor (RBD for receptor binding domain). In particular, FIG. 2 a describes all the deletion mutants designed from EnvSU. FIG. 2 b 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 Env197, Env168 and Env144 mutants and the binding defect of the Env69-317, Env169-317 and Env117 mutants.

FIG. 3 a 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. FIG. 3 b 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 (antiTM-EnvW).

FIG. 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 Reticuloendotheliosis Virus), BAEV (Baboon endogenous virus (strain M7)), SRV1 (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).
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 FIG. 1 a.

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 production in a serum-free medium and filtered on 0.45 μm membranes in order to remove the cellular debris. 20 μl 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 XChASCT1 constitutively expressing the hASCT2 (XChASCT2) or hASCT1 (XChASCT1) 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 PiTl receptor.
The cells were washed in PBS and harvested by detaching with 0.02% versene in PBS. A total of 10⁶cells 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 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, XChASCT1, 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 hASCT1 and hASCT2 (FIG. 1 b). 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 PiT1 receptor, but did not bind to the TE671RD cells blocked for the hASCT2 receptor (FIG. 1 c). 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, Env197, Env168, Env169-317, Env117 and Env144 (FIG. 2 a) were transfected into the HEK293T cells by precipitation with calcium phosphate. The conditions for production and analysis of the proteins are identical to those detailed in example 1.
EnvSU, Env69-317 and Env197 proteins were correctly expressed in soluble form. Using the XC cells constitutively expressing hASCT2, the inventors demonstrated that the Env197 protein was capable of binding to the receptor expressed at the surface of the cells like the SU subunit (1-317). Thus, the first 176 residues of the mature SU subunit (and therefore lacking its signal peptide) were sufficient for binding to the surface of the cells expressing the hASCT2 receptor. The deletion of the 21-68 region resulted in a loss of binding to the receptor also indicating its involvement in the receptor binding domain (RBD). On the other hand, the truncated Env168 protein showed a lower capacity for binding to the hASCT2 receptor.
In order to obtain the equivalent quantities in the supernatants between the various truncated proteins, the inventors fused two smaller domains of the N-terminal region of SU (Env117 and Env144) to the C-terminal region of the SU subunit (Env169-317), the latter domain not binding to hASCT2. The level of expression of the Env117 and Env144 proteins was similar and the proteins were expressed in soluble form. The binding test showed that only the Env144 protein was capable of binding to the cells expressing the hASCT2 receptor. The absence of binding of the Env117 protein to the surface of the cells indicated the loss of at least one determinant of binding inside the 117-144 region.
Consequently, the boundaries of the domains for interaction of the W envelope with its receptor are defined by amino acids 21 to 144.
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 A E: 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)

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 Env144 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 Env144 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

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 μg/μl of total proteins.
Immunization of Mice with an Extract of Cellular Lysate
The same mice first of all received an injection of 10 μg of cellular lysate by the intraperitoneal route followed by a booster injection of 2×100 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 μg 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 1F11B10 were thus obtained. The monoclonal antibodies 2H1H8 and 12C7A3 are directed against the nonglycosylated N-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 1F11B10 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 1F11B10 monoclonal antibodies (dilution 1/50th). Next, they are reinoculated at equal concentration (0.4×10⁵cells/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 2×10⁵cells per well and the co-culture is continued for 24 h. An XGal (5-bromo-4-chloro-3-indolyl-β-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-Grünwald and Giemsa solutions (MERCK) performed according to the suppliers' recommendations. The fusion observed corresponds to a fusion “from within”, that is to say a 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.

TABLE 1

Antibody	23A5	6A2B2	2H1H8	1F11B10	12C7A3

100 ng	261	231	130	223	ND
500 ng	217	273	73	210	11

ND: not determined

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 1F11B10) 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 CRL8805), in culture in DMEM medium (Gibco Invitrogen 41966-029) supplemented with South American serum, were respectively transfected, with the aid of the LipofectAMINE PLUSH 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 (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 409are 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 PLUS™ kit:

1 Precomplexing of the DNA

- mixing of 750 μl of medium not supplemented with the abovementioned DNAs in a 15 ml falcon tube (reference 2096), that is 2 μl of 409 or 3 μl of 410 or 3 μl LQMV;
- stirring under vortex of PLUS reagent and adding 20 μl thereof to the DNA solution;
- stirring under vortex immediately 10 seconds at 1400 rpm;
- incubation 15 minutes at room temperature.

2 Preparation of the Cells

- replacement with 5 ml of nonsupplemented medium.

3 Dilution of the LipofectAMINE

In a tube for a dish, mix 30 μl of LipofectAMINE Reagent with 750 μl of nonsupplemented medium.

4 Complexing of the DNA

Mixing of 780 μl of dilute LipofectAMINE and 772 μl of solution of precomplexed DNA (total: 1552 μl); 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 μl 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), ⅕th 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 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 (3×2=6 mice)
the cells transfected with the three types of plasmid (DNAs 409, 410 and LQMV) with the monoclonal antibody 2H1H8 (3×2=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 syncytia

	Lines	S1* count	S2* count	Mean

409 control	19	22	20.5
409 + 2H1H8	3	4	3.5
410 control	8	11	9.5
410 + 2H1H8	2	3	2.5
LQMV control	8	5	6.5
LQMV + 2H1H8	1	1	1

S1* and S2* = mouse 1 and mouse 2

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:
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<0.02)
Env versus control LQMV: Chi-2=9.83 (p<0.002)
Env versus the two controls (410 and LQMV): Chi-2=7.69 (p<0.01)
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<0.01). Subsequent analysis, by staining, of the specificity of the effects therefore merely confirm the specificity of the effect obtained in vivo in the presence of the Env protein, thereby validating the animal model of the in vivo study of syncytia whose fusion was induced by HERV-W Env.
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,
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<0.001)
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 0.45 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

1 IP (Intraperitoneal) Inoculation of the XChASCT1, XChASCT2 Cells and Control Cells XChASCT—into SCID Mice
Injection into mice of ⅕th 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 μl of supernatant with 10 μl of antibody ( 1/100th 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 (1×10⁶cells per point, that is ⅕th 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 μl 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).
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 ( 1/20th 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 μl 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) Nice:

Groups of 2 mice are inoculated with:
each type of cell (expressing the 2 types of receptor hASCT1 and hASCT2 and one not expressing the receptor hASCT—as a control) with no antibody (3×2=6 mice) the three types of cell with the Env protein and the monoclonal antibody 2H1H8 (3×2=6 mice).

4) Results

The results by immunofluorescence (IF) reading with a microscope are presented in Table 3 below:

TABLE 3

IF reading: number of fluorescent cells/total
number of cells (NF/NT) in the same field

Lines	NF/NT	Mean

XC control
1/18, 0/10	1/28
XC + 2H1H8	0/20	0/20
XChASCT1 control	12/40, 3/12, 9/25	24/77
XChASCT1 + 2H1H8	1/25, 0/18, 1/30	2/73
XChASCT2 control	8/22, 15/35	23/57
XChASCT1 + 2H1H8	1/45	1/45

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 hASCT1 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 hASCT1 (control hASCT1) 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 hASCT1 (control hASCT1) 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:
control hASCT1: 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 hASCT1 versus hASCT−: Chi-2=8.62 (p<0.01)
Env+grafts hASCT2 versus HASCT−: Chi-2=12.53 (p<0.001)
Env+grafts hASCT1 versus env+grafts hASCT2: Chi-2=1.21 (difference not significant).
The cells expressing the hASCT1 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 hASCT1 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 hASCT1: 24 positives counted on average out of 77 cells
Env+grafts hASCT1+monoclonal antibody 2H1H8: 2 positives counted on average out of 73 cells.
Env+grafts hASCT1 alone versus injection antibody 2H1H8: Chi-2=21.14 (p<0.001)
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<0.001).
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), SRV1 (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.

Claims

1. A peptide domain necessary for an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the peptide domain comprising:

an N-terminus motif having an amino acid sequence selected from the group consisting of: SEQ ID No. 1 to SEQ ID No. 29,

a C-terminus motif having an amino acid sequence selected from the group consisting of: SEQ ID No. 30 to SEQ ID No. 40, and

at least one motif between the N-terminus and the C-terminus, and having an amino acid sequence selected from the group consisting of: SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 73.

2. A nucleotide sequence encoding a peptide domain as defined in claim 1.

3. An epitope derived from the peptide domain of claim 1, wherein the epitope induces an immune response against a virus belonging to the HERV-W interference group.

4. A nucleotide sequence encoding an epitope as defined in claim 3.

5. An expression vector comprising:

the nucleotide sequence of claim 2, and

elements necessary for expressing the nucleotide sequence.

6. A microorganism or host cell transformed with at least one expression vector as defined in claim 5.

7. An antibody directed against a peptide domain as defined in claim 1.

8. A method of inhibiting, preventing and/or treating in an animal an infection caused by a virus belonging to an HERV-W interference group, the method comprising:

administering to the animal, in an effective amount, a composition comprising as an active substance at least one peptide domain of claim 1 and elements necessary for a constitutive and/or inducible expression of said peptide domain.

9. A pharmaceutical composition comprising, as an active substance, at least one peptide domain of claim 1 and elements necessary for a constitutive and/or inducible expression of said peptide domain, and a pharmaceutically acceptable vehicle.

10. A diagnostic composition for detecting and/or quantifying a virus belonging to an HERV-W interference group, and/or for quantifying an immune response against said virus, the composition comprising at least one peptide domain of claim 1.

11. A method for detecting and/or quantifying virus belonging to an HERV-W interference group in a biological sample taken from an individual liable to be infected by said virus, the method comprising:

contacting said biological sample with at least one antibody of claim 7 under conditions allowing formation of a complex between the virus and the antibody, and

detecting and/or quantifying the formation of said complex.

12. A method of in vitro screening of a virus belonging to an HERV-W interference group in a biological sample or specimen, the method comprising:

contacting the composition of claim 10 with the biological sample or specimen, and

determining whether an immune response to the virus is produced.

13. A method of inhibiting an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising contacting the peptide domain of claim 1 with said hASCT receptor.

14. A method of identifying chemical or biological molecules whose interaction with all or part of the peptide domain of claim 1 blocks an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising:

contacting at least one chemical or biological molecule with the peptide domain of claim 1; and

detecting whether interaction occurs between the envelope of the virus belonging to the HERV-W interference group and the hASCT receptor.

15. A method of generating antibodies which block an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising inoculating an animal or cultured cells with the peptide domain of claim 1, thereby inducing the animal or cultured cells to generate antibodies directed against the peptide domain of claim 1.

16. A method of determining a peptide domain necessary for an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising:

identifying a nucleotide and/or peptide sequence of a precursor envelope of said virus,

excluding a signal part of said sequence,

detecting an amino acid motif having an amino acid sequence corresponding to SEQ ID No. 43, and

excluding a C-terminus in the amino acid sequence between 15 and 25 amino acid positions after the amino acid motif having an amino acid sequence corresponding to SEQ ID No. 43.

17. A peptide domain determined by the method of claim 16.

18. The method of claim 8, wherein the animal is a human.

19. An antibody produced by the method of claim 15.

20. A method of producing a peptide domain necessary for an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising:

transforming a microorganism or a host cell with at least one expression vector of claim 5,

culturing the transformed microorganism or host cell, and

recovering the peptide domain produced by the microorganism or host cell.

21. An expression vector comprising:

the nucleotide sequence of claim 4, and

elements necessary for expressing the nucleotide sequence.

22. A microorganism or host cell transformed with at least one expression vector of claim 21.

23. A method of producing a peptide domain necessary for an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising:

transforming a microorganism or host cell with at least one expression vector of claim 21,

culturing the transformed microorganism or host cell, and

recovering the peptide domain produced by the microorganism or host cell.

24. An antibody directed against the epitope of claim 3.

25. A method of inhibiting, preventing and/or treating in an animal an infection caused by a virus belonging to an HERV-W interference group, the method comprising:

administering to the animal, in an effective amount, a composition comprising as an active substance at least one epitope of claim 3 and elements necessary for a constitutive and/or inducible expression of said epitope.

26. The method of claim 25, wherein the animal is a human.

27. A method of inhibiting, preventing and/or treating in an animal an infection caused by a virus belonging to an HERV-W interference group, the method comprising:

administering to the animal, in an effective amount, a composition comprising as an active substance at least one antibody of claim 7.

28. The method of claim 27, wherein the animal is a human.

29. A method of inhibiting, preventing and/or treating in an animal an infection caused by a virus belonging to an HERV-W interference group, the method comprising:

administering to the animal, in an effective amount, a composition comprising as an active substance at least one nucleotide sequence of claim 2.

30. The method of claim 29, wherein the animal is a human.

31. A method of inhibiting, preventing and/or treating in an animal an infection caused by a virus belonging to an HERV-W interference group, the method comprising:

administering to the animal, in an effective amount, a composition comprising as an active substance at least one nucleotide sequence of claim 4.

32. The method of claim 31, wherein the animal is a human.

33. A pharmaceutical composition comprising, as an active substance, at least one epitope of claim 3 and elements necessary for a constitutive and/or inducible expression of said epitope, and a pharmaceutically acceptable vehicle.

34. A pharmaceutical composition comprising, as an active substance, at least one nucleotide sequence of claim 2, and a pharmaceutically acceptable vehicle.

35. A pharmaceutical composition comprising, as an active substance, at least one nucleotide sequence of claim 4, and a pharmaceutically acceptable vehicle.

36. A pharmaceutical composition comprising, as an active substance, at least one antibody of claim 7, and a pharmaceutically acceptable vehicle.

37. A diagnostic composition for detecting and/or quantifying a virus belonging to an HERV-W interference group, and/or for quantifying an immune response against said virus, the composition comprising at least one epitope of claim 3.

38. A diagnostic composition for detecting and/or quantifying a virus belonging to an HERV-W interference group, and/or for quantifying an immune response against said virus, the composition comprising at least one nucleotide sequence of claim 2.

39. A diagnostic composition for detecting and/or quantifying a virus belonging to an HERV-W interference group, and/or for quantifying an immune response against said virus, the composition comprising at least one nucleotide sequence of claim 4.

40. A diagnostic composition for detecting and/or quantifying a virus belonging to an HERV-W interference group, and/or for quantifying an immune response against said virus, the composition comprising at least one antibody of claim 7.

41. A method of inhibiting an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising contacting the epitope of claim 3 with said hASCT receptor.

42. A method of inhibiting an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor, the method comprising contacting the antibody of claim 7 with said envelope.