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WO1992018630A1 - Compound comprising a peptide sequence neutralizing the infection by human cytomegalovirus (hcmv) and composition containing such compound - Google Patents

Compound comprising a peptide sequence neutralizing the infection by human cytomegalovirus (hcmv) and composition containing such compound Download PDF

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Publication number
WO1992018630A1
WO1992018630A1 PCT/FR1992/000351 FR9200351W WO9218630A1 WO 1992018630 A1 WO1992018630 A1 WO 1992018630A1 FR 9200351 W FR9200351 W FR 9200351W WO 9218630 A1 WO9218630 A1 WO 9218630A1
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compound
infection
hcmv
sequence
compound according
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PCT/FR1992/000351
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French (fr)
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Jean-Michel Alonso
François Xavier DERAMOUDT
Sylvie Monnet
Catherine David
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Fondation Nationale De Transfusion Sanguine
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Publication of WO1992018630A1 publication Critical patent/WO1992018630A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • COMPOUND COMPRISING A PEPTIDE SEQUENCE NEUTRALIZING HUMAN CYTOMEGALOVIRUS (HCMV) INFECTION AND COMPOSITION COMPRISING SAME
  • the present invention relates to the use of a peptide sequence of human 02 microglobulin ( ⁇ 2m) or an ohgopeptide included in this sequence, or a molecule including all or appearing of this sequence, capable of neutralizing infection by HCMV.
  • HCMV is a Herpes virus implicated in various syndromes ranging from the classic "disease of cytomegalic inclusions" of the newborn, to infectious and immunopathological complications of blood transfusions and tissue transplants, or of various latrogenic or acquired immunodeficiency syndromes, including AIDS (Merigan. TC. And Resta, S 1990, Rev. infect. Dis, 12: s 693).
  • Murine models, using MCMV. have helped to confirm the hypotheses resulting from the observation of human pathology: the decompensation of latent infection would be secondary to the immune deficiency of nitrogen, predominantly altering cell-mediated immunity (T lymphocytes and monocytes - macrophages), but secondary triggering of tissue damage (in particular interstitial pneumonia) would result from an immunopathological reaction (probably the action of cytotoxic lymphocytes against infected cells expressing viral "neoantigens”) (Shanley et al. 1982, J. Infect. Dis. 146: 388).
  • MHCI major histocompatibility class I complex
  • the inventors evaluated different synthetic peptides, the sequence of which reproduces portions of the ⁇ 2m. for their ability, when combined with HCMV, to inhibit infection of human cells and murine transfectants expressing human ⁇ 2m. The inhibitory power of HCMV infection of cells has been demonstrated for one of these peptides.
  • the present invention relates to a compound endowed in particular with an inhibitory activity of the infection of human cells by the cytomegalovirus, characterized in that it contains at least the sequence Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr- Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val (522V) or a fragment of said sequence having inhibitory activities.
  • the compound according to the present invention may contain other peptide sequences and / or sequences of a non-peptide nature as will be described below. It should be understood that the amino acid sequence thus described may include other functionalities, in particular glycosylations, which are covered by the preceding definition.
  • the compound according to the present invention can be obtained by various techniques, in particular by chemical synthesis or obtained in the form of a recombinant molecule expressed by a prokaryotic or eukaryotic host.
  • a peptide containing the neutralizing sequence can also be obtained by chemical or enzymatic cleavages of the native ⁇ 2m, using agents such as: mild acid hydrolysis, hydroxylamine, CNBr, BNPS or NCS / urea, NB5 , NZB, Armillaria mellea protease, chymotrypsin, clostripain, endopeptidase Lys C, pancreatic elastase, post-proline cleavage enzyme. trypsin. staphylococcus protease V8 for example.
  • Ganciclovir and Foscarnet are the most active. Acyclovir has not been shown to work. Ganciclovir has the disadvantage of causing toxic effects for granulocytes and Foscarnet is nephrotoxic. Protocols combining polyclonal anti-HC MV immunoglobins with Ganciclovir and making it possible to reduce the dosage of the antiviral would have made it possible to limit the mortality rate from HCMV infection.
  • Anti-HCMV immunogiobulins have a beneficial effect in delaying and limiting the onset of infection after renal transplant.
  • Attenuated vaccines injected into kidney transplant recipients, would have a beneficial effect in reducing the severity of the infection.
  • One of the therapeutic approaches which is proposed in the present invention is the use of the mechanism for neutralizing viral infection by inhibiting the attachment of HCMV to its presumed receptor, ⁇ 2m, by saturating the virus with a " "peptide lure corresponding to one of the domains of the native ⁇ 2m.
  • the compound according to the invention can be used in the form of the peptide sequence or in a more elaborate form.
  • protective elements and / or carrier molecules may be elements ends resistant to peptidases: hydroxy, non-natural acid and / or amino acid for example, or protective elements of the PEG, immunoglobulin and / or albumin type or fragments derived from these products.
  • This compound can also be combined in the form of particles, nanoparticles, liposomes or the like, in particular inert and biodegradable.
  • the present invention also relates to anti-idiotypes of antibodies directed against the sequence described above.
  • an antibody directed against this peptide can mimic its structure and conformation its binding site to HCMV.
  • An anti-idiotype against this antibody may have the same inhibitory functions for HCMV infection as the neutralizing peptide and therefore be used as a substitute for the latter.
  • this peptide can be used as a vectoring agent for molecules active against HCMV (antiviral agents, interferon for example).
  • the subject of the invention is pilot drugs, that is to say comprising all or part of molecules with anti-viral activity coupled with the compounds and / or peptides according to the invention.
  • the antiviral agents that can be used are more particularly analogs of antiviral nucleosides such as Acyclovir or Ganciclovir or Foscarnet.
  • the coupling methods are known and depend on the products in question. It is now possible to carry out couplings on the NH 2 and / or COOH fractions of the peptides according to the invention.
  • the present invention also relates to the pharmaceutical composition
  • the pharmaceutical composition comprising at least one compound according to the invention, either as an active principle or as a vectorizing agent for molecules active against HCMV.
  • the compounds according to the present invention may be in any dosage form suitable for the mode of administration.
  • the dosages used obviously depend on the type of infection and its stage and must be adapted to the patient's condition.
  • Figure 1 inhibitory effect of S22V, on human fibroblasts MRC 5 and murine J27, expressing human ⁇ 2 microglobulin.
  • Figure 2 dose-dependence of the neutralizing effect of 522V on human MRC5 fibroblasts exposed to HCMV.
  • Figure 3 Effect of pre-incubation of synthetic peptides, derived from ⁇ 2m, with MRC5 cells, on their sensitivity to HCMV infection.
  • Figure 4 Absence of competition between the native zm and the peptide S22V for the neutralizing power of the latter on the HCMV.
  • Figure 5 Neutralizing effect of peptide S22V compared to that of therapeutic anti-CMV IgG.
  • the HCMV AD 169 strain was provided to us by S. Michelson (Institut Pasteur, Paris).
  • the viral inocuium is obtained from supernatants of MRC5 cells (ATCC CCL 171) cultivated in MEM medium (Gibco) supplemented with fetal calf serum (SVF) at a concentration of 10% final, and infected for a period which causes 90% cytopathic effect.
  • the HCMC stock is kept at -80 ° C.
  • the cells are first cultivated in a 24-well plate (Falcon) in MEM, 10% FCS, until the formation of a monolayer cell mat. Twenty four hours before infection the culture medium is replaced by MEM without additive.
  • the viral inoculum is mixed v / v with each product and incubated for 1 h at 37 ° C. before being brought into the presence of the target cells.
  • the proven products are:
  • a control synthetic peptide (NEOSYSTEM) (D 20K) having no sequence homology with (32m and whose amino acid sequence is:
  • a human anti-HCMV monoclonal IgG2 (JM Seigneurin, CHRU de Grenoble) was also tested in our trials.
  • the culture medium is removed after 1 8 hours of incubation of the infected cells.
  • the cells are washed twice with 0.1 M NaCI in phosphate buffer (PBS).
  • the cells are then fixed in 90% acetone for 1 5 minutes at + 4 ° C.
  • HC MV infection is detected by revelation of immediately early antigens (IEA) with a murine monoclonal antibody IgG l, (Ref: E 13 Biosoft) itself revealed by murine anti IgG immunoconjugate (H + L) the peroxidase (Pasteur Diagnosis).
  • the results presented express the average of the number of infectious units detected according to the number of cell nuclei stained by the immunoperoxidase revealed by the addition of diaminobenzidine tetrahydrochloride then H2O2.
  • the human fibroblasts MRC 5 and the murine fibroblasts 327 expressing the human 2 m are sensitive to infection by HCMV AD 169, while the control murine fibroblasts transfected with the TK gene are insensitive.
  • These results confirm the receptor role for (32m for HCMV.
  • Pre-incubation with the various preparations at a concentration of 200 ug / ml shows that the S22V peptide inhibits infection of MRC5 and 327.
  • the C225 peptide does not seems to inhibit in this trial only MRC5 infection
  • the peptide D20K was omitted from the MRC5 infection trial.
  • FIG. 2 shows the results obtained with three concentrations of the products tested against HCMV on MRC5 cells. It is confirmed that the S22V peptide brought into contact with HCMV is neutralizing and that this effect is dose-dependent.
  • Example 3 Specificity of the inhibitory effect of the peptide S22V Three synthetic peptides representing domains of the ⁇ 2m were therefore tested for their HCMV inhibitory activities in vitro. It therefore appears that a synthetic peptide of 21 aa (S-22-V) representing the amino acid sequence 61-82 of human ⁇ 2m has an inhibitory power against infection by the HCMV strain of reference AD-169, reference target cells (human fibroblasts MRC5) as well as murine fibroblasts transfected with the human ⁇ 2m gene.
  • FIG. 3 indicates that the pre-incubation of the MRC5 cells with the different peptide preparations before infection with CMV does not induce a dose-dependent neutralizing effect (the binding of the peptide to the virus is necessary for the inhibition of the infection).
  • the proposed mode of action is the inhibition of the attachment of the "coated" HCMV by the peptide to its site of attachment to the target cell.
  • AD-169 HCMV virus
  • S-22-V peptide a competition trial between S-22-V and the native ⁇ 2m was carried out.
  • a neutralization experiment was carried out for a peptide concentration of 200 ⁇ g / ml (8 ⁇ 10 -2 M) in the presence of 2, 20 and 200 mg / ml of ⁇ 2m (1.8 ⁇ 10 -4 , 1.8 ⁇ 10 -3 and 1.8 ⁇ 10 -2 M).
  • Native ⁇ 2m has no inhibitory effect on the effect of the peptide in vitro.
  • FIG. 5 indicates that the S-22-V peptide at a concentration of 200 ⁇ g / ml (8 ⁇ 10 -2 M) has an inhibitory power equivalent to that of a preparation of intravenous anti-CMV immunoglobulins (BioTransfusion : lot no. 52 01 00 10) at a dilution of 1/10 equivalent to 10 IU / ml of anti-CMV.
  • IU / ml level of anti-CMV antibody determined by ELISA method, by reference to the international standard).
  • Figure 3 Experimental neutralization of infection, in vitro, by HCMV of human MRC5 fibroblasts, pre-incubated in the presence of 2, 20 and 200 ⁇ g / ml of the peptides S22V, E23M and C22S; Results corresponding to the average of the measurements made on four wells ⁇ standard deviation.
  • Figure 4 Neutralization power of S-22-V on AD-169 in the presence of native ⁇ 2m at different concentrations.
  • Figure 5 Comparative neutralizing power between the peptide S-22-V at 200 ⁇ g / ml and the specific intravenous Ig G anti-CMV diluted to 1/10, 1/100 and 1/1000.

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Abstract

The present invention relates to a compound comprising a peptide inhibiting the infection of human cells by the human cytomegalovirus, characterized in that the peptide contains at least the sequence: Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val or a fragment of said sequence. The invention also relates to a composition comprising at least one compound as hereabove defined, usable particularly for therapeutical purposes.

Description

COMPOSE COMPRENANT UNE SEQUENCE PEPTIDIQUE NEUTRALISANT L'INFECTION PAR LE CYTOMEGALOVIRUS HUMAIN (HCMV) ET COMPOSITION LE COMPORTANT COMPOUND COMPRISING A PEPTIDE SEQUENCE NEUTRALIZING HUMAN CYTOMEGALOVIRUS (HCMV) INFECTION AND COMPOSITION COMPRISING SAME
La présente invention concerne l'utilisation d'une séquence peptidique de la 02 microglobuline ( β2m) humaine ou un ohgopeptide compris dans cette séquence, ou une molécule incluant tout ou parue de cette séquence, capable de neutraliser l'infection par le HCMV.  The present invention relates to the use of a peptide sequence of human 02 microglobulin (β2m) or an ohgopeptide included in this sequence, or a molecule including all or appearing of this sequence, capable of neutralizing infection by HCMV.
Le HCMV est un Herpès-virus incriminé dans différents syndromes allant de la classique "maladie des inclusions cytomégaliques" du nouveau-né, aux complications infectieuses et immunopathoiogiques des transfusions sanguines et greffes tissulaires, ou de différents syndromes immunodéficitaires latrogènes ou acquis, dont le SIDA (Merigan. TC. and Resta, S 1990, Rev. infect. Dis, 12 : s 693).  HCMV is a Herpes virus implicated in various syndromes ranging from the classic "disease of cytomegalic inclusions" of the newborn, to infectious and immunopathological complications of blood transfusions and tissue transplants, or of various latrogenic or acquired immunodeficiency syndromes, including AIDS (Merigan. TC. And Resta, S 1990, Rev. infect. Dis, 12: s 693).
Le portage latent chez l'adulte chniquemént sain varierait de 40 à 100 % selon les contrées (Ho, M, 1990, Rev. Infect. Dis, 1 2 : S 7 01 ).  Latent carriage in healthy adults generally varies from 40 to 100% depending on the country (Ho, M, 1990, Rev. Infect. Dis, 1 2: S 7 01).
Les mécanismes pathogéniques qui régissent la décompensauor, de l'infection latente lors de l'immunosuppression, ainsi que les comohcations immunopathologiques, après greffe allogénique, sont méconnus.  The pathogenic mechanisms which govern decompensator, of latent infection during immunosuppression, as well as the immunopathological comohcations, after allogenic transplantation, are little known.
Leur identification est entravée par l'absence de modèle expérimental chez l'animal de laboratoire, du fait de la stricte spécificaté du HCMV pour l'homme.  Their identification is hampered by the absence of an experimental model in laboratory animals, due to the strict specificity of HCMC for humans.
Des modèles murins, utilisant le MCMV. ont perms de conforter les hypothèses résultant de l'observation de la pathologie humaine : la décompensation de l'infection latente serait secondaire au déficit immunitaire latrogène, altérant de manière prédominante l'immunité à médiation cellulaire (lymphocytes T et monocytes - macrophages), mais le déclenchement secondaire de lésions tissulaires (notamment la pneumonie interstitielle) résulterait d'une réaction immunopathologique (probablement l'action des lymphocytes cytotoxiques contre les cellules infectées exprimant des "néoantigènes" viraux) (Shanley et coll. 1982, J. Infect. Dis. 146 : 388).  Murine models, using MCMV. have helped to confirm the hypotheses resulting from the observation of human pathology: the decompensation of latent infection would be secondary to the immune deficiency of nitrogen, predominantly altering cell-mediated immunity (T lymphocytes and monocytes - macrophages), but secondary triggering of tissue damage (in particular interstitial pneumonia) would result from an immunopathological reaction (probably the action of cytotoxic lymphocytes against infected cells expressing viral "neoantigens") (Shanley et al. 1982, J. Infect. Dis. 146: 388).
Cependant les différences génomiques et phénotypiques entre le MCMV et Je HCMV ne permettent pas de validation immunopathologique directement transposahle à l'homme, à partir des données du modèle moins La compréhension des mécanismes d'interaction entre le HCMV et la cellule-hôte a bénéficié de deux découvertes récentes qui suggèrent que le virus pourrait se fixer à la membrane cellulaire sur le complexe majeur d'histocompatibilité de classe I (MHCI) : le part que le HCMV he la β 2 microglobuline, chaîne légère du MHCI. tant dans les produits pathologiques, qu'expérimentalement (Grundy, JE et coll, 1987. J. Gen. Virol. 68 : 793) et, corrélativement, l'identification d'une séquence génomique virale codant potentiellement pour une protéine analogue du MHCI (Beck, S, and Barrel G., 1988, Nature, 331 : 269). However, the genomic and phenotypic differences between MCMV and I HCMV do not allow immunopathological validation directly transposed to humans, from model data less Understanding the mechanisms of interaction between HCMV and the host cell has benefited from two recent discoveries which suggest that the virus could bind to the cell membrane on the major histocompatibility class I complex (MHCI): the share that HCMV he β 2 microglobulin, light chain of MHCI. both in pathological products and experimentally (Grundy, JE et al, 1987. J. Gen. Virol. 68: 793) and, correlatively, the identification of a viral genomic sequence potentially coding for a protein analogous to MHCI ( Beck, S, and Barrel G., 1988, Nature, 331: 269).
Ces données ont conduit les inventeurs à év aluer, e: a démontrer, le rôle de la β2m humaine en tant que récepteur pour le HCMV en utilisant des fibroblastes murins, normalement non permissifs au HCMV . transfectés avec le gène de la β2m humaine, comme cellules-cmies.  These data have led the inventors to assess, and demonstrate: the role of human β2m as a receptor for HCMV using murine fibroblasts, normally non-permissive to HCMV. transfected with the human β2m gene, like cells-cmies.
A partir de ces résultats et dans un des aspects de la présente invention, les inventeurs ont évalué différents peptides synthétiques, dont la séquence reproduit des portions de la β2m. pour leur capacité, une fois associés au HCMV, à inhiber l'infection des cellules humaines et des transfectants murins exprimant la β2m humaine. Le pouvoir inhibiteur de l'infection des cellules par le HCMV a été démontré pour l'un de ces peptides.  On the basis of these results and in one aspect of the present invention, the inventors evaluated different synthetic peptides, the sequence of which reproduces portions of the β2m. for their ability, when combined with HCMV, to inhibit infection of human cells and murine transfectants expressing human β2m. The inhibitory power of HCMV infection of cells has been demonstrated for one of these peptides.
La présente invention concerne un composé doté notamment d'une activité inhibitrice de l'infection de cellules humaine s par le cytomégalovirus, caractérisé en ce qu'il contient au moins la séquence Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val (522V) ou un fragment de ladite séquence présentant des activités inhibitrices.  The present invention relates to a compound endowed in particular with an inhibitory activity of the infection of human cells by the cytomegalovirus, characterized in that it contains at least the sequence Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr- Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val (522V) or a fragment of said sequence having inhibitory activities.
Le composé selon la présente invention peut comporter d'autres séquences peptidiques et/ou des séquences de nature non peptidique comme cela sera décrit ci-après. Il doit être compris que la séquence en amino-acides ainsi décrite peut comporter d'autres fonctionnalités, des glycosylations notamment, qui sont couvertes par la définition précédente. Le composé selon la présente invention peut être obtenu par différentes techniques, notamment par synthèse chimique ou obtenu sous forme de molécule recombinante exprimée par un hôte procaryote ou eucaryote. The compound according to the present invention may contain other peptide sequences and / or sequences of a non-peptide nature as will be described below. It should be understood that the amino acid sequence thus described may include other functionalities, in particular glycosylations, which are covered by the preceding definition. The compound according to the present invention can be obtained by various techniques, in particular by chemical synthesis or obtained in the form of a recombinant molecule expressed by a prokaryotic or eukaryotic host.
Un peptide contenant la séquence neutralisante peut être également obtenu par coupures chimiques ou enzymatiques de la β2m native, en utilisant des agents tels que : l'hydrolyse acide ménagée, l'hydroxylamine, le CNBr, le BNPS ou le NCS/urée, le NB5, le NZB, la protease d'Armillaria mellea, la chymotrypsine, la clostripaîne, l'endopeptidase Lys C, l'élastase pancréatique, l'enzyme de clivage post-proline. la trypsine. la protease V8 du staphylocoque par exemple.  A peptide containing the neutralizing sequence can also be obtained by chemical or enzymatic cleavages of the native β2m, using agents such as: mild acid hydrolysis, hydroxylamine, CNBr, BNPS or NCS / urea, NB5 , NZB, Armillaria mellea protease, chymotrypsin, clostripain, endopeptidase Lys C, pancreatic elastase, post-proline cleavage enzyme. trypsin. staphylococcus protease V8 for example.
Ces composés selon l'invention présentent une approche thérapeutique originale de la lutte contre le HCMV.  These compounds according to the invention present an original therapeutic approach to the fight against HCMV.
En effet l'approche thérapeutique actuelle en matière de prévention et de traitement des infections à HCMV implique l'utilisation de molécules antivirales et/ou immunothérapeutiques (Mérigan. TC, and Resta. S., 1990, Rev. Infect. Dis, 12 : S 693).  In fact, the current therapeutic approach in the prevention and treatment of HCMV infections involves the use of antiviral and / or immunotherapeutic molecules (Mérigan. TC, and Resta. S., 1990, Rev. Infect. Dis, 12: S 693).
Parmi les agents antiviraux administrés dans un but curatif à des patients immunodéprimés souffrant d'une infection à HC MV. le Ganciclovir et le Foscarnet seraient les plus actifs. L'Acyclovir n'a pas d'efficacité démontrée. Le Ganciclovir présente l'inconvénient d'entraîner des effets toxiques pour les granulocytes et le Foscarnet est néphrotoxique. Des protocoles associant des îmmunoglobines polyclonales anti-HC MV au Ganciclovir et permettant de diminuer la posologie de l'antiviral auraient permis de limiter le taux de mortalité par infection à HCMV.  Among the antiviral agents administered for curative purposes to immunocompromised patients suffering from HC MV infection. Ganciclovir and Foscarnet are the most active. Acyclovir has not been shown to work. Ganciclovir has the disadvantage of causing toxic effects for granulocytes and Foscarnet is nephrotoxic. Protocols combining polyclonal anti-HC MV immunoglobins with Ganciclovir and making it possible to reduce the dosage of the antiviral would have made it possible to limit the mortality rate from HCMV infection.
Les immunogiobulines anti-HCMV auraient un effet bénéfique en retardant et limitant la survenue de l'infection après greff e rénale.  Anti-HCMV immunogiobulins have a beneficial effect in delaying and limiting the onset of infection after renal transplant.
Cet effet n'est pas évident dans le cas de greffe allogénique de moelle osseuse. Des vaccins atténués, injectés à des transplantés rénaux, auraient un effet bénéfique en diminuant la gravité de l'infection.  This effect is not evident in the case of allogeneic bone marrow transplantation. Attenuated vaccines, injected into kidney transplant recipients, would have a beneficial effect in reducing the severity of the infection.
Ces différents produits thérapeutiques nécessitent d'être évalués dans des essais cliniques complets, pour leur intérêt en traitement et/ou prévention incluant leurs effets sur la suppression d u portage asymptomatique, qui semble être la source et le réservoir unique de l'infection. These different therapeutic products need to be evaluated in complete clinical trials, for their value in treatment and / or prevention including their effects on the suppression of asymptomatic carriage, which seems to be the source and the sole reservoir of infection.
Les bilans des essais cliniques ne permettent pas d'attriouer d'effet satisfaisant aux molécules thérapeutiques administrées aux patients immmunodeficients ou transplantés. La quête de nouveaux produits efficaces et dénués d'effets secondaires est essentielle.  The results of clinical trials do not allow to attribute a satisfactory effect to the therapeutic molecules administered to immunodeficient or transplanted patients. The quest for new effective products with no side effects is essential.
L'une des approches thérapeutiques qui est proposée dans la présente invention est l'utilisation du mécanisme de neutralisation de l'infection virale par inhibition de l'attachement du HCMV à son récepteur présumé, la β 2m, en saturant le virus par un "leurre" peptidique correspondant à un des domaines de la β2m native.  One of the therapeutic approaches which is proposed in the present invention is the use of the mechanism for neutralizing viral infection by inhibiting the attachment of HCMV to its presumed receptor, β 2m, by saturating the virus with a " "peptide lure corresponding to one of the domains of the native β2m.
Une telle approche, par "détournement" de récepteur a déjà été tentée avec succès dans d'autres modèles d'infection virale : le CD4 pour HIVI et l'ICAMl pour le Rhinovirus (W hite, JM, and Littman, D., 1989. Cell, 56 : 725).  Such a receptor "hijacking" approach has already been successfully attempted in other models of viral infection: CD4 for HIVI and ICAMl for Rhinovirus (W hite, JM, and Littman, D., 1989 Cell, 56: 725).
C'est précisément l'un des buts de la présente invention que de détourner le virus de son récepteur en utilisant l'un des composés définis précédemment.  It is precisely one of the aims of the present invention to divert the virus from its receptor by using one of the compounds defined above.
Le composé selon l'invention peut être utilisé sous forme ce la séquence peptidique ou bien sous une forme plus élaborée.  The compound according to the invention can be used in the form of the peptide sequence or in a more elaborate form.
Ainsi, afin d'augmenter la durée de vie du compose selon l'invention, lors de son administration, on peut prévoir de le coupler chimiquement ou physiquement avec des éléments protecteurs et/ou des molécules porteuses, il pourra s'agir d'éléments d'extrêmités résistants aux peptidases : hydroxy, acide et/ou amino acide non naturel par exemple, ou bien des éléments protecteurs du type PEG, immunoglobuline et/ou albumine ou bien des fragments dérivés de ces produits. Ces technologies de protection sont également connues et ont déjà été appliquées à différents peptides.  Thus, in order to increase the lifetime of the compound according to the invention, during its administration, provision may be made to chemically or physically couple it with protective elements and / or carrier molecules, it may be elements ends resistant to peptidases: hydroxy, non-natural acid and / or amino acid for example, or protective elements of the PEG, immunoglobulin and / or albumin type or fragments derived from these products. These protection technologies are also known and have already been applied to various peptides.
Ce composé peut également être associé sous des formes de particules, nanoparticules, liposomes ou autres, en particulier inertes et biodégradables. La présente invention concerne également les anti-idiotypes des anticorps dirigés contre la séquence décrite précédemment. This compound can also be combined in the form of particles, nanoparticles, liposomes or the like, in particular inert and biodegradable. The present invention also relates to anti-idiotypes of antibodies directed against the sequence described above.
En effet, un anticorps dirigé contre ce peptide peut mimer dans sa structure et conformation son site de liaison au HCMV. U n anti-idiotype contre cet anticorps peut avoir les mêmes fonctions inhibitrices de l'infection par le HCMV que le peptide neutralisant et donc être employé comme substitut de celui-ci.  Indeed, an antibody directed against this peptide can mimic its structure and conformation its binding site to HCMV. An anti-idiotype against this antibody may have the same inhibitory functions for HCMV infection as the neutralizing peptide and therefore be used as a substitute for the latter.
Outre son pouvoir inhibiteur, ce peptide peut être utilisé comme agent de vectorisation de molécules actives contre le HCMV (agents antiviraux, interféron par exemple).  In addition to its inhibitory power, this peptide can be used as a vectoring agent for molecules active against HCMV (antiviral agents, interferon for example).
Ainsi, l'invention a pour objet des médicaments pilotes c'est-à-dire comportant tout ou partie de molécules à activité anti-virale couplées avec les composés et/ou les peptides selon l'invention. Les anti-viraux utilisables sont plus particulièrement des analogues de nucleosides anti-viraux type Acyclovir ou Ganciclovir ou Foscarnet. Les méthodes de couplage sont connues et dépendent des produits en cause. Il est maintenant possible d'effectuer des couplages sur les fractions NH 2 et/ou COOH des peptides selon l'invention. Thus, the subject of the invention is pilot drugs, that is to say comprising all or part of molecules with anti-viral activity coupled with the compounds and / or peptides according to the invention. The antiviral agents that can be used are more particularly analogs of antiviral nucleosides such as Acyclovir or Ganciclovir or Foscarnet. The coupling methods are known and depend on the products in question. It is now possible to carry out couplings on the NH 2 and / or COOH fractions of the peptides according to the invention.
La présente invention concerne également la composition pharmaceutique comportant au moins un composé selon l'invention, soit a titre de principe actif soit comme agent de vectorisation de molécules actives contre le HCMV.  The present invention also relates to the pharmaceutical composition comprising at least one compound according to the invention, either as an active principle or as a vectorizing agent for molecules active against HCMV.
Les composés selon la présente invention peuvent se présenter sous une forme galénique quelconque appropriée au mode d'administration. Les dosages utilisés dépendent évidemment du type d'infection et de son stade et doivent être adaptés à l'état du patient.  The compounds according to the present invention may be in any dosage form suitable for the mode of administration. The dosages used obviously depend on the type of infection and its stage and must be adapted to the patient's condition.
Les exemples ci-après sont destinés à mettre en valeur d'autres avantages et caractéristiques de la présente invention sans pour autant être limitatifs.  The examples below are intended to highlight other advantages and characteristics of the present invention without however being limiting.
Les figures 1 , 2, 3, 4 et 5 suivantes illustrent l'invention.  The following Figures 1, 2, 3, 4 and 5 illustrate the invention.
Figure 1 : effet inhibiteur de S22V, sur les fibroblastes humains M R C 5 et murins J27, exprimant la β 2 microglobuline humaine. Figure 2 : dose-dépendance de l'effet neutralisant de 522V sur les fibroblastes humains MRC5 exposés au HCMV. Figure 1: inhibitory effect of S22V, on human fibroblasts MRC 5 and murine J27, expressing human β 2 microglobulin. Figure 2: dose-dependence of the neutralizing effect of 522V on human MRC5 fibroblasts exposed to HCMV.
Figure 3 : Effet de la pré-incubation des peptides synthétiques, dérivés de la β2m, avec les cellules MRC5, sur la sensibilité de celles-ci à l'infection par le HCMV.  Figure 3: Effect of pre-incubation of synthetic peptides, derived from β2m, with MRC5 cells, on their sensitivity to HCMV infection.
Figure 4 : Absence de compétition entre la zm native et le peptide S22V pour le pouvoir neutralisant de celui-ci sur le HCMV.  Figure 4: Absence of competition between the native zm and the peptide S22V for the neutralizing power of the latter on the HCMV.
Figure 5 : Effet neutralisant du peptide S22V comparé à celui des IgG anti-CMV thérapeutiques.  Figure 5: Neutralizing effect of peptide S22V compared to that of therapeutic anti-CMV IgG.
Exemple 1 : Infections virales  Example 1: Viral infections
La souche de HCMV AD 169 nous a été fournie par S. Michelson (Institut Pasteur, Paris). L'inocuium viral est obtenu à partir de surnageants de cellules MRC5 (ATCC CCL 171) cultivées en milieu MEM (Gibco) additionné de sérum de veau f étal (SVF) à la concentration de 10 % final, et infectées pour une période qui provoque 90 % d'effet cytopathique. Le stock de HCMV est conservé à -80°C. The HCMV AD 169 strain was provided to us by S. Michelson (Institut Pasteur, Paris). The viral inocuium is obtained from supernatants of MRC5 cells (ATCC CCL 171) cultivated in MEM medium (Gibco) supplemented with fetal calf serum (SVF) at a concentration of 10% final, and infected for a period which causes 90% cytopathic effect. The HCMC stock is kept at -80 ° C.
Outre les cellules fibroblastiques humaines MRC5, nous avons éprouvé le pouvoir infectieux du HCMV, seul ou pré-incubé avec divers produits, pour des fibroblastes murins (L) transfectés soit avec le gène de la thymidine-kinase (témoin) ou soit avec le gène de la β2m humaine (cellules J27). Ces transfectants nous ont été fournis par M. Pla (INSER M L 93, Hôpital St Louis, Paris).  In addition to human MRC5 fibroblast cells, we tested the infectious power of HCMV, alone or pre-incubated with various products, for murine fibroblasts (L) transfected either with the thymidine kinase gene (control) or with the gene. human β2m (J27 cells). These transfectants were provided to us by M. Pla (INSER M L 93, Hôpital St Louis, Paris).
Les cellules sont d'abord cultivées en plaque à 24 puits (Falcon) en MEM, 10 % SVF, jusqu'à formation d'un tapis celluiaire monocouche. Vingt quatre heures avant l'infection le milieu de culture est remplacé par du MEM sans additif.  The cells are first cultivated in a 24-well plate (Falcon) in MEM, 10% FCS, until the formation of a monolayer cell mat. Twenty four hours before infection the culture medium is replaced by MEM without additive.
Au moment de l'infection le milieu est remplacé par la suspension virale et la plaque est centrifugée à 2 000 × g pendant 45 minutes. L'excès de virus est ensuite éliminé et les cellules sont mises en culture en MEM sans additif. Les plaques sont incubées à 37°C en air CO2 à 5 % pendant 18 heures. Exemple 2 : Essais de neutralisation At the time of infection, the medium is replaced by the viral suspension and the plate is centrifuged at 2000 × g for 45 minutes. The excess virus is then eliminated and the cells are cultured in MEM without additive. The plates are incubated at 37 ° C in 5% CO2 air for 18 hours. Example 2: Neutralization tests
1. Matériel et méthodes Pour évaluer le pouvoir neutralisant des différentes préparations contre le HCMV, l'inoculum viral est mélangé v/v à chaque produit et incubé pendant 1 h à 37°C avant d'être mis en présence des cellules cibles. 1. Materials and methods To assess the neutralizing power of the various preparations against HCMV, the viral inoculum is mixed v / v with each product and incubated for 1 h at 37 ° C. before being brought into the presence of the target cells.
Les produits éprouvés sont :  The proven products are:
. La 2m humaine (SEROTEC)  . The human 2m (SEROTEC)
. Un peptide synthétique (NEOSYSTEM) témoin ( D 20K ) ne présentant aucune homologie de séquence avec la (32m et dont la séquence en acides aminés est :  . A control synthetic peptide (NEOSYSTEM) (D 20K) having no sequence homology with (32m and whose amino acid sequence is:
Asp-Pro-Arg-Val-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly-Ser-Ser-Ser-Gly-Thr-Val-Lys-.  Asp-Pro-Arg-Val-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly-Ser-Ser-Ser-Gly-Thr-Val-Lys-.
. Trois peptides synthétiques (NEOSYSTEM) représentant une partie de la séquence peptidique de la β2m humaine (Cunningham. B A.. Wang, JL., Berggard, I., and Peterson, PA, 1973, Biochemistry, 1 2 : 48 1 1 ) : le peptide E23.M : Glu-Tyr-Ala-Cys-Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile-Val-Cys-Trp-Asp-Arg-Met, le peptide C22S : Cys-Ile-Glu-Val-Asp-Leu-Leu-Lys-Asn-Gly-Glu-Arg-Ile-GIu-Lys-Val-Glu-His-Ser-Asp-Leu-Ser et le peptide 522 : Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val.  . Three synthetic peptides (NEOSYSTEM) representing part of the peptide sequence of human β2m (Cunningham. B A .. Wang, JL., Berggard, I., and Peterson, PA, 1973, Biochemistry, 1 2: 48 1 1) : the peptide E23.M: Glu-Tyr-Ala-Cys-Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile-Val-Cys-Trp-Asp-Arg-Met , the C22S peptide: Cys-Ile-Glu-Val-Asp-Leu-Leu-Lys-Asn-Gly-Glu-Arg-Ile-GIu-Lys-Val-Glu-His-Ser-Asp-Leu-Ser and the peptide 522: Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val.
. Une IgG2 monoclonale humaine anti-HCMV (JM Seigneurin, CHRU de Grenoble) a également été éprouvée dans nos essais.  . A human anti-HCMV monoclonal IgG2 (JM Seigneurin, CHRU de Grenoble) was also tested in our trials.
Le milieu de culture est éliminé après les 1 8 heures d'incubation des cellules infectées. Les cellules sont lavées deux fois avec du NaCI 0, 1 5 M en tampon phosphate (PBS). Les cellules sont ensuite f ixées en 90 % d'acétone pendant 1 5 minutes à +4°C. L'infection par le HC MV est détectée par révélation des antigènes immédiatement précoces (IEA ) a vec un anticorps monoclonal murin IgG l, (Réf : E 13 Biosoft) lui-même revélé par immunoconjugué anti IgG murine (H+L) marqué à la péroxv dase (Diagnostic Pasteur). Les résultats présentés expriment la moyenne du nombre d'unités infectieuses détectées d'après le nombre de noyaux cellulaires colorés par l'immunopéroxydase révélée par adjonction de diaminobenzidine tetrahydrochlorure puis H2O2. 2. Résultats The culture medium is removed after 1 8 hours of incubation of the infected cells. The cells are washed twice with 0.1 M NaCI in phosphate buffer (PBS). The cells are then fixed in 90% acetone for 1 5 minutes at + 4 ° C. HC MV infection is detected by revelation of immediately early antigens (IEA) with a murine monoclonal antibody IgG l, (Ref: E 13 Biosoft) itself revealed by murine anti IgG immunoconjugate (H + L) the peroxidase (Pasteur Diagnosis). The results presented express the average of the number of infectious units detected according to the number of cell nuclei stained by the immunoperoxidase revealed by the addition of diaminobenzidine tetrahydrochloride then H2O2. 2. Results
Comme le montre le figure 1, les fibroblastes humains MRC 5 et les fibroblastes murins 327 exprimant la 2m humaine sont sensibles a l'infection par HCMV AD 169, alors que les fibroblastes murins témoins transfectés avec le gène TK sont insensibles. Ces résultats confirment le rôle de récepteur de la (32m pour le HCMV. La pré-incubation avec les différentes préparations à la concentration de 200 ug/ml montre que le peptide S22V inhibe l'infection des MRC5 et des 327. Le peptide C225 ne semble inhiber dans cet essai que l'infection des MRC5. Dans cette expérience le peptide D20K a été omis de l'essai d'infection αes MRC5. As shown in FIG. 1, the human fibroblasts MRC 5 and the murine fibroblasts 327 expressing the human 2 m are sensitive to infection by HCMV AD 169, while the control murine fibroblasts transfected with the TK gene are insensitive. These results confirm the receptor role for (32m for HCMV. Pre-incubation with the various preparations at a concentration of 200 ug / ml shows that the S22V peptide inhibits infection of MRC5 and 327. The C225 peptide does not seems to inhibit in this trial only MRC5 infection In this experiment the peptide D20K was omitted from the MRC5 infection trial.
La figure 2 montre les résultats obtenus avec trois concentration des produits éprouvés contre le HCMV sur cellules MRC5. Il se confirme que le peptide S22V mis en présence du HCMV est neutralisant et que cet effet est dose-dépendant.  FIG. 2 shows the results obtained with three concentrations of the products tested against HCMV on MRC5 cells. It is confirmed that the S22V peptide brought into contact with HCMV is neutralizing and that this effect is dose-dependent.
Exemple 3 : Spécificité de l'effet inhibiteur du peptide S22V Trois peptides synthétiques représentant des domaines de la β2m ont donc été testés pour leurs activités inhibitrices du HCMV in vitro. Il apparaît donc qu'un peptide synthétique de 21 aa (S-22-V) représentant la séquence en amino-acide 61-82 de la β2m humaine a un pouvoir inhibiteur de l'infection par la souche HCMV de référence AD- 169, de cellules cibles de référence (fibroblastes humain MRC5) ainsi que de fibroblastes murins transfectés par le gène de la β2m humaine. Example 3: Specificity of the inhibitory effect of the peptide S22V Three synthetic peptides representing domains of the β2m were therefore tested for their HCMV inhibitory activities in vitro. It therefore appears that a synthetic peptide of 21 aa (S-22-V) representing the amino acid sequence 61-82 of human β2m has an inhibitory power against infection by the HCMV strain of reference AD-169, reference target cells (human fibroblasts MRC5) as well as murine fibroblasts transfected with the human β2m gene.
La figure 3 indique que la pré-incubation des cellules MRC5 avec les différentes préparations de peptide avant l'infection par CMV n'induit pas d'effet neutralisât dose-dépendant (La fixation du peptide sur le virus est nécessaire à l'inhibition de l'infection). FIG. 3 indicates that the pre-incubation of the MRC5 cells with the different peptide preparations before infection with CMV does not induce a dose-dependent neutralizing effect (the binding of the peptide to the virus is necessary for the inhibition of the infection).
Le mode d'action proposé est l'inhibition de l'attachement du HCMV "enrobé" par le peptide à son site de fixation sur la cellule cible. Pour évaluer la spécificité et l'affinité de l'interaction entre le virus HCMV (AD- 169) etle peptide S-22-V, un essai de compétition entre S-22-V et la β2m native a été effectué. Une expérience de neutralisation a été réalisée pour une concentration de peptide de 200 μg / ml (8 × 10-2 M) en présence de 2, 20 et 200 mg / ml de β2m (1,8 × 10-4, 1,8 × 10-3 et 1,8 × 10-2 M). The proposed mode of action is the inhibition of the attachment of the "coated" HCMV by the peptide to its site of attachment to the target cell. To assess the specificity and affinity of the interaction between the HCMV virus (AD-169) and the S-22-V peptide, a competition trial between S-22-V and the native β2m was carried out. A neutralization experiment was carried out for a peptide concentration of 200 μg / ml (8 × 10 -2 M) in the presence of 2, 20 and 200 mg / ml of β2m (1.8 × 10 -4 , 1.8 × 10 -3 and 1.8 × 10 -2 M).
Les résultats, présentés dans la figure 4, montrent que quelle que soit la dose de β2m ajoutée, le pouvoir neutralisant du peptide S-22-V est constant The results, presented in FIG. 4, show that whatever the dose of β2m added, the neutralizing power of the peptide S-22-V is constant
Il n'y a donc pas d'interférence a priori de la β2m soluble sur l'effet neutralisant du peptide S-22-V, même à des concentrations de β2m cent fois plus élevées que la concentration physiologique dans le plasma (Howard MR, Me Verry BA, Cooper EH. A longitudinal study of sérum beta 2-microglobulin levels in haemophilia. Clin. Lab. Haemat. 1988, 10, 427-434). There is therefore no a priori interference of soluble β2m on the neutralizing effect of the peptide S-22-V, even at concentrations of β2m one hundred times higher than the physiological concentration in plasma (Howard MR, Me Verry BA, Cooper EH. A longitudinal study of serum beta 2-microglobulin levels in haemophilia. Clin. Lab. Haemat. 1988, 10, 427-434).
La β2m native n'a aucun effet inhibiteur sur l'effet du peptide in vitro. Native β2m has no inhibitory effect on the effect of the peptide in vitro.
Cette spécificité de l'effet du peptide comme inhibiteur de l'infection par le HCMV a été confirmée : - Le S-22-V est incapable de neutraliser l'infection des cellules MRC 5 par le virus de la polio (souche SABIN). - Cette inhibition de l'infection n'est pas due à la synthèse d'un autre inhibiteur par les cellules, tel l'interféron α ou le Fibroblast Growth Factor (FGF). This specificity of the effect of the peptide as an inhibitor of HCMV infection has been confirmed: - S-22-V is unable to neutralize the infection of MRC 5 cells by the polio virus (strain SABIN). - This inhibition of the infection is not due to the synthesis of another inhibitor by the cells, such as interferon α or the Fibroblast Growth Factor (FGF).
Enfin, la figure 5 indique que le peptide S-22-V à une concentration de 200 μg / ml (8 × 10-2 M) a un pouvoir inhibiteur équivalent à celui d'une préparation d'immunoglobulines anti-CMV intraveineuses (BioTransfusion : lot n°52 01 00 10) à la dilution 1 / 10 équivalent à 10 UI / ml d' anti-CMV. (UI / ml = taux en anticorps anti-CMV déterminé par méthode ELISA, par référence à l'étalon international). Légendes : Finally, FIG. 5 indicates that the S-22-V peptide at a concentration of 200 μg / ml (8 × 10 -2 M) has an inhibitory power equivalent to that of a preparation of intravenous anti-CMV immunoglobulins (BioTransfusion : lot no. 52 01 00 10) at a dilution of 1/10 equivalent to 10 IU / ml of anti-CMV. (IU / ml = level of anti-CMV antibody determined by ELISA method, by reference to the international standard). Legends:
Figure 3 : Neutralisation expérimentale de l'infection, in vitro, par HCMV de fibroblastes humains MRC5, pré-incubées en présence de 2, 20 et 200 μg/ml des peptides S22V, E23M et C22S ; Résultats correspondant à la moyenne des mesures faites sur quatre puits ± écart type. Figure 3: Experimental neutralization of infection, in vitro, by HCMV of human MRC5 fibroblasts, pre-incubated in the presence of 2, 20 and 200 μg / ml of the peptides S22V, E23M and C22S; Results corresponding to the average of the measurements made on four wells ± standard deviation.
Figure 4 : Pouvoir de neutralisation de S-22-V sur AD-169 en présence de β2m native à différentes concentrations. Figure 4: Neutralization power of S-22-V on AD-169 in the presence of native β2m at different concentrations.
Figure 5 : Pouvoir neutralisant comparatif entre le peptide S-22-V à 200 μg/ml et les Ig G intraveineuses spécifiques anti-CMV diluées au 1 / 10, 1/100 et 1/1000. Figure 5: Comparative neutralizing power between the peptide S-22-V at 200 μg / ml and the specific intravenous Ig G anti-CMV diluted to 1/10, 1/100 and 1/1000.

Claims

REVENDICATIONS
1. Composé doté notamment d'une activité inhibitrice de l'infection de cellules humaines par le cytomégalovirus humain, caractérisé en ce qu'il comprend au moins la séquence 1. Compound endowed in particular with an inhibitory activity of the infection of human cells by the human cytomegalovirus, characterized in that it comprises at least the sequence
Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val  Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val
ou un fragment de ladite séquence présentant des propriétés inhibitrices. or a fragment of said sequence having inhibitory properties.
2. Composé selon la revendication 1 caractérisé en ce que il est couplé avec un élément protecteur et/ou des molécules porteuses.  2. Compound according to claim 1 characterized in that it is coupled with a protective element and / or carrier molecules.
3. Composé selon l'une des revendications 1 à 2 caractérisé en ce qu'il comporte un fragment destiné à améliorer sa résistance aux peptidases.  3. Compound according to one of claims 1 to 2 characterized in that it comprises a fragment intended to improve its resistance to peptidases.
4. Composé selon 1 et 3 caractérisé en ce que la séquence est associée à une molécule naturelle, telle que l'albumine, ou une immunoglobuline.  4. Compound according to 1 and 3 characterized in that the sequence is associated with a natural molecule, such as albumin, or an immunoglobulin.
5. Composé selon 1 à 4 caractérisé en ce que la séquence est associée à un liposome ou à une nanoparticule inerte et biodégradable.  5. Compound according to 1 to 4, characterized in that the sequence is associated with a liposome or with an inert and biodegradable nanoparticle.
6. Composé selon 1 à 5 caractérisé en ce qu'il est obtenu par fractionnement chimique ou enzymatique de la fi microglobuline ou par synthèse chimique.  6. Compound according to 1 to 5, characterized in that it is obtained by chemical or enzymatic fractionation of the microglobulin or by chemical synthesis.
7. Composé selon 1 à 6 caractérisé en ce qu'il est exprimé sous forme d'une molécule recombinante chez un hôte procaryote ou eucaryote.  7. Compound according to 1 to 6, characterized in that it is expressed in the form of a recombinant molecule in a prokaryotic or eukaryotic host.
8. Composé selon l'une des revendications 1 à 7 caractérisé en ce que il comporte en plus de la séquence, tout ou partie d'une molécule à activité anti-virale.  8. Compound according to one of claims 1 to 7 characterized in that it comprises, in addition to the sequence, all or part of a molecule with anti-viral activity.
9. Composition comportant au moins un composé tel que défini dans l'une des revendications 1 à 8.  9. Composition comprising at least one compound as defined in one of claims 1 to 8.
10. Composition pharmaceutique selon 9 comportant au moins un composé tel que défini dans l'une des revendications 1 à 8. 10. Pharmaceutical composition according to 9 comprising at least one compound as defined in one of claims 1 to 8.
1 1. Composition pharmaceutique selon 10 utilisée à titre de thérapeutique ou de prophylaxie contre une affection à cytomegalovirus. 1 1. Pharmaceutical composition according to 10 used as therapy or prophylaxis against a cytomegalovirus disease.
12. Anti-idiotype contre l'anticorps d'un composé selon l'une des revendications 1 à 11, utilisable à titre de vaccin.  12. Anti-idiotype against the antibody of a compound according to one of claims 1 to 11, usable as a vaccine.
PCT/FR1992/000351 1991-04-18 1992-04-17 Compound comprising a peptide sequence neutralizing the infection by human cytomegalovirus (hcmv) and composition containing such compound WO1992018630A1 (en)

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Publication number Priority date Publication date Assignee Title
FR2735984A1 (en) * 1995-06-30 1997-01-03 Inst Nat Sante Rech Med VACCINE AGAINST INFECTIOUS AGENTS HAVING AN INTRACELLULAR PHASE, COMPOSITION FOR THE TREATMENT AND PREVENTION OF HIV INFECTIONS, ANTIBODIES AND DIAGNOSTIC METHOD
WO1997002344A3 (en) * 1995-06-30 1997-03-06 Inst Nat Sante Rech Med Vaccine for infectious agents, composition for treating and preventing hiv infections
US6113902A (en) * 1995-06-30 2000-09-05 Institut National De La Santa Et De La Recherche Medicale (Inserm) Immunogenic compositions comprising peptides from β-2-microglobulin

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