Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
  • C07K16/4208—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
  • C07K16/4241—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
  • C07K16/4258—Immunoglobulins [IG], 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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• 1991
  • 1991-04-18 FR FR9104787A patent/FR2675508A1/fr active Granted
• 1992
  • 1992-04-17 WO PCT/FR1992/000351 patent/WO1992018630A1/fr not_active Ceased

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