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

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

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US20090148454A1
US20090148454A1 US12/087,893 US8789307A US2009148454A1 US 20090148454 A1 US20090148454 A1 US 20090148454A1 US 8789307 A US8789307 A US 8789307A US 2009148454 A1 US2009148454 A1 US 2009148454A1
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herv
virus
envelope
peptide domain
interference group
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Francois Mallet
Guy Oriol
Valerie Cheynet
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Biomerieux SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10061—Methods of inactivation or attenuation
    • C12N2740/10063—Methods of inactivation or attenuation by chemical treatment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus

Definitions

  • 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.
  • HERVs Human endogenous retroviruses
  • 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).
  • SU surface subunit gp50
  • TM transmembrane subunit
  • 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).
  • ASCT family a receptor-transporter of amino acids of the ASCT family
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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:
  • 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.
  • 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.
  • 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.
  • ⁇ is an integer between 3 and 18.
  • 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.
  • X a , X b , X c or the amino acids at positions 3, 4 and 5 of SEQ ID Nos. 30 to 40 are a glycine, X d or 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 f 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, X g or 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.
  • ⁇ is an integer equal to 20.
  • X 2 or the amino acid Xaa at position 3 of SEQ ID No. 41 is an amino acid chosen from asparagine, threonine, glutamic acid, histidine, X 3 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; X 4 or the amino acid Xaa at position 5 of SEQ ID No. 41 is an amino acid chosen from tyrosine, threonine, alanine, X 5 or the amino acid Xaa at position 6 of SEQ ID No. 41 is an amino acid chosen from glutamine, arginine, threonine, X 6 or the amino acid Xaa at position 7 of SEQ ID No. 41 is an amino acid chosen from leucine, glutamine, glutamic acid.
  • X 7 or the amino acid Xaa at position 2 of SEQ ID No. 42 is an amino acid chosen from proline, threonine, arginine and asparagine;
  • X 8 or 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, X 10 or the amino acid Xaa at position 5 of SEQ ID No.
  • SEQ ID No. 42 is lysine or is deleted;
  • X 11 or the amino acid xaa at position 6 of SEQ ID No. 42 is an amino acid chosen from lysine, valine, isoleucine, leucine,
  • X 12 or the amino acid Xaa at position 7 of SEQ ID No. 42 is an amino acid chosen from glycine, asparagine;
  • 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 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.
  • the invention also relates to a nucleotide sequence encoding a peptide domain according to the invention above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an animal 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)′ 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.
  • 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).
  • scFv fragment (“single chain Fragment variable”) is then used because it consists of a single polypeptide chain.
  • 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.
  • 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.
  • 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
  • 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.
  • the active substance for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, rectal or transdermal administration, 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.
  • the active substance may be used in creams, ointments, lotions, eyedrops.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • a peptide domain according to the invention of HERV-W when 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.
  • 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.
  • 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.
  • 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.
  • a retroviral vector pseudotyped by the envelope of interest and expressing a detectable marker (LacZ) 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 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).
  • a marker for example, LacZ
  • HERV-W env transiently or stably expressing the envelope of interest
  • 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:
  • X a , X b , X c is an amino acid which is glycine
  • X d is an amino acid chosen from proline and valine
  • X e is an amino acid chosen from glutamine, leucine and threonine
  • X f is an amino acid chosen from lysine, threonine, methionine and glutamine
  • X g is an amino acid chosen from alanine, lysine, isoleucine, threonine and valine.
  • 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 Dyrl ⁇ v Bendtsen, Henrik Nielsen, Gunnar von Hiejne and S ⁇ ren 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.
  • FIG. 1 illustrates the phenotypical characteristics and properties of soluble recombinant proteins derived from Env-W.
  • 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).
  • 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).
  • 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:
  • 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).
  • FIG. 1 a 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.
  • 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 ⁇ m membranes in order to remove the cellular debris. 20 ⁇ l 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.
  • 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 10 6 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 flow cytometry.
  • PBA PBS and 0.5% sodium azide
  • RGSH6, Quiagen an anti-histidine monoclonal antibody
  • the cells were washed once with PBA and incubated with a secondary antibody coupled to fluorescein isocyanate for 1
  • 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.
  • 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.
  • the SU subunit is capable of binding to two receptors hASCT1 and hASCT2 ( FIG. 1 b ).
  • this protein was tested in relation to human cells TE671 and derived cells TE671RD and TE671galv.
  • 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.
  • 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).
  • 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).
  • the truncated Env168 protein showed a lower capacity for binding to the hASCT2 receptor.
  • 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.
  • 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.
  • 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)).
  • 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.
  • the use of an antibody not directed against the RBD did not adversely affect its binding to the hASCT2 receptor.
  • mice 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 ⁇ g of DNA were first performed for each mouse followed by a booster with two injections of 4 ⁇ g 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.
  • 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 ⁇ g/ ⁇ l of total proteins.
  • mice first of all received an injection of 10 ⁇ g of cellular lysate by the intraperitoneal route followed by a booster injection of 2 ⁇ 100 ⁇ g of cellular lysate by the intraperitoneal route.
  • Another injection was performed, by the intravenous route, with 22 ⁇ g 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.
  • 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 ⁇ g/ ⁇ l of total proteins and an Env AS protein as negative control at a concentration of 13.3 ⁇ g/ ⁇ l 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.
  • 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).
  • Epithelioid-carcinoma-indicating human cells Hela, ATCC CCL-2
  • Epithelioid-carcinoma-indicating human cells Hela, ATCC CCL-2
  • An XGal (5-bromo-4-chloro-3-indolyl- ⁇ -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)).
  • 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 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.
  • mice Groups of 2 mice are inoculated with:
  • 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.
  • 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.
  • 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.
  • 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.
  • Soluble protein supernatant filtered on 0.45 ⁇ m containing the soluble protein (293T cells transfected with the plasmid 460 (envelope-spacer-His6).
  • Antibody monoclonal antibody 2H1H8 (IgG, 5.50 mg/ml).
  • XChASCT2 cellular clone XC (ATCC CCL-165, rat cells) expressing the hASCT2 receptor.
  • DMEM medium Gibco Invitrogen 41966-029 with South American serum.
  • mice of 1 ⁇ 5th of the flask at 70% confluence in a volume of 2 ml.
  • IP intraperitoneal inoculation of the proteins alone or with the antibody into mice transplanted with the cells (1 ⁇ 10 6 cells per point, that is 1 ⁇ 5th of a confluent dish 100 mm in diameter).
  • the pellet is taken up in 100 ⁇ l 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.
  • PBA buffer PBS with 2% fetal calf serum and 0.1% sodium azide
  • Pellet taken up in 500 ⁇ l of PBA, maintained at +4° C., and analyzed by FACS.
  • mice Groups of 2 mice are inoculated with:
  • NF/NT 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
  • the reality of the cells that bound the Env protein to their hASCT1 or hASCT2 receptor was then verified by cytofluorometric analysis.
  • 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)
  • 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.
  • 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.
  • 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).
  • 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 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.

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