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WO2008009777A1 - Glycoproteines de hsv ayant une activité inductrice de la migration cellulaire médiée par la chimiokine - Google Patents

Glycoproteines de hsv ayant une activité inductrice de la migration cellulaire médiée par la chimiokine Download PDF

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Publication number
WO2008009777A1
WO2008009777A1 PCT/ES2007/070139 ES2007070139W WO2008009777A1 WO 2008009777 A1 WO2008009777 A1 WO 2008009777A1 ES 2007070139 W ES2007070139 W ES 2007070139W WO 2008009777 A1 WO2008009777 A1 WO 2008009777A1
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hsv
protein
amino acid
acid sequence
chemokine
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Abel Viejo Borbolla
Antonio Alcami Pertejo
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C07K14/035—Herpes simplex virus I or II
    • 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
    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Biomedicine and biotechnology Development of active therapeutic principles. Development of pharmaceutical compositions for the treatment of human diseases that present with alterations of the migration of cells of the immune system.
  • Chemokines are a family of 8-10 kDa basic cytokines that induce leukocyte migration and infiltration of infected or damaged tissues (Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature, 392: 565-568) playing a fundamental role in defense against pathogens and in inflammatory diseases. Chemokines are located on the cell surface through their interaction with glycosaminoglycans (GAGs), a necessary interaction for the correct presentation of chemokines to leukocytes and their function in vivo (Cinamon, G., Shinder, V. and Alón, R.
  • GAGs glycosaminoglycans
  • chemokine system is complex, with more than 50 human chemokines identified classified into four classes (C, CC, CXC and CX3C) and about 20 receptors.
  • C, CC, CXC and CX3C The critical role of chemokines in anti-viral defense and in the immune response in general is reflected in the discovery of many viral mechanisms to modulate chemokine activity (Alcami A. (2003). Viral mimicry of cytokines, chemokines and their receptors Nat. Rev.
  • vCKBP viral chemokine-binding protein
  • glycoprotein G present in some alpha herpesviruses has that ability (Bryant, NA, Davis-Poynter, N., Vanderplasschen, A., and Alcami, A. (2003) Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum Chemokine binding proteins EMBO J., 22: 833-846; Costs B, Ruiz-Arguello MB, Bryant NA, Alcami A, Vanderplasschen A. (2005) Both soluble and membrane-anchored forms of Felid herpesvirus 1 glycoprotein G function as a broad-spectrum chemokine-binding protein J. Gen. Virol. 86: 3209-3214).
  • herpesvirus alpha subfamily have a variable host range and the ability to establish latent infections primarily but not exclusively in sensory ganglia (Roizman, B. and Pellet, PE (2001) The family herpesviridae: and introduction. In Knipe , DM and Howley, PM (eds.), Fielols Virology. Lippincott Williams and Wilkins, Philadelphia, VoI. 2, pp. 2381-2397).
  • This subfamily includes animal pathogens such as equine herpesviruses 1 and 3 (EHV-I and EHV-3) and bovine herpesviruses 1 and 5
  • BHV-I and BHV-5 human pathogens such as HSV-I and HSV-2, which replicate in oral and genital mucosa and varicella zoster virus (VZV).
  • VZV varicella zoster virus
  • the function of glycoprotein G (gG) was unknown until it was discovered that the gG encoded by alpha herpesviruses constitutes a new family of viral proteins capable of binding to Chemokines (Bryant, NA, Davis-Poynter, N., Vanderplasschen, A., and Alcami, A.
  • the gG of EHV-I, EHV-3, BHV-I and BHV-5 has binding activity to a wide range of chemokines with high affinity, inhibits the interaction of chemokines with cell receptors and their activity of inducing cell migration, to despite lacking sequence similarity with cell chemokine receptors.
  • chemokines play a critical role in activating the inflammatory response, inducing the migration of immune cells to infected or damaged tissues, gG's ability to inhibit chemokines postulated the anti-inflammatory role of gG.
  • no such activity was observed in the gG of HSV-I and HSV -2 - which has very low sequence similarity with the gGs of equine and bovine viruses - with the chemokines used in this invention
  • feline herpesvirus is able to bind chemokines (B. Costes, MB Ruiz-Argüello, NA Bryant, A. Alcami and A. Vanderplasschen. 2005. Both soluble and membrane-anchored forms of feline herpesvirus 1 glycoprotein G function as a broad spectrum chemokine binding protein. J Gen. Virol. 86, 3209-3214).
  • HSV-I and HSV-2 are important human pathogens. HSV-I infects and replicates, usually in the gold-labial and ocular mucosa. HSV-2 replicates in the genital mucosa.
  • Herpes simplex 1 and 2 viruses (HSV-I and HSV-2) are human pathogens with a high prevalence in the population, so that HSV-I in adults is between 60 and 90% depending on the geographical area of the world. The Reactivation of both viruses causes the production of viral particles with the appearance of lesions that have a great clinical impact. HSV-I is the cause of corneal keratitis that can cause blindness, meningitis and is the main viral cause of encephalitis due to its neurotropic character.
  • HSV-I and HSV-2 can cause encephalitis if the infection takes place in the central nervous system.
  • Herpetic encephalitis is a focal brain process located in the frontotemporal and parietal areas of the brain.
  • Subacute encephalitis associated with HSV has been identified in 2% of the cases studied in patients with acquired immunodeficiency syndrome (AIDS).
  • HSV poses a risk to newborns since infection during the third trimester of gestation and / or childbirth can lead to disseminated disease, failure of multiple organs, including the central nervous system, and death of the fetus or neonate.
  • mortality exceeds 80% in disseminated disease and 50% in those patients who have symptoms only in the CNS.
  • HSV pathology A new aspect of HSV pathology that has acquired special relevance in recent years is the association of HSV infections with neurodegenerative diseases such as Alzheimer's.
  • HSV-I and HSV-2 the mechanisms employed by HSV-I and HSV-2 in the modulation and evasion of the immune system, determinants of viral pathogenesis, are poorly understood so that their compression may allow the development of new therapeutic approaches.
  • An object of the present invention is the use of a protein compound derived from the HSV glycoprotein (gG) inducer of chemokine activity, hereinafter use of a gG protein of the present invention, for the preparation of a medicament or pharmaceutical composition for the treatment of a disease or disorder caused by a deficit of chemokine-mediated cell migration.
  • gG HSV glycoprotein
  • a particular embodiment of the invention is the use of the gG protein where the use comprises the use of one or more amino acid sequences belonging to the following group: a) an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-I or HSV-2, b) an amino acid sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) a sequence of amino acids comprising any sequence belonging to a), b) and c).
  • Another object of the present invention is a nucleotide sequence characterized in that it is an extracellular fragment of the HSV-I gG gene, preferably constituted by SEQ ID NO1, which codes for the HSV-I gG-ls form or by the SEQ ID NO3, which codes for the gG-2s form of HSV-2 (Example 1).
  • Another object of the present invention is a peptide or protein derived or fragment of the HSV gG, either of the HSV-I or HSV-2 type, inducing chemokine-mediated cell migration.
  • Another realization Particular of the invention is an extracellular fragment protein of the HSV-I gG, preferably constituted by SEQ ID N02, gG-ls protein form of HSV-I or SEQ ID NO4, gG-2s protein form of HSV -2 (Example 1).
  • Another object of the present invention is a pharmaceutical composition or medicament for the treatment of diseases, disorders or pathologies caused or associated with a deficit of cell migration mediated by chemokines, hereinafter pharmaceutical composition of the present invention, which comprises a protein compound Chemokine-mediated cell migration inducer of the invention, in therapeutically effective amount together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.
  • a particular embodiment of the invention is a pharmaceutical composition of the invention in which the gG protein or peptide belongs to the following group: a) an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-I or HSV-2, b) an amino acid sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) an amino acid sequence comprising a sequence any belonging to a), b) and c).
  • Another object of the invention is the use of the pharmaceutical composition of the invention, hereinafter use of the pharmaceutical composition of the invention, in a method of treatment or prophylaxis of a mammal, preferably a human being, affected by a disease, disorder or pathology that involves alterations in chemokine-mediated cell migration consisting of the administration of said therapeutic composition in adequate dose that allows the recovery of the cell migration capacity, preferably of cells of the immune system.
  • Another object of the present invention is a method of identifying compounds useful for the treatment of infections caused by HSV characterized in that the HSV gG protein or an extracellular fragment thereof is used, either of the HSV-I type or HSV-I, as a therapeutic target.
  • the present invention is based on the fact that the inventors have observed that the glycoprotein (gG) of the human viruses HSV-I and HSV-2, of great clinical relevance, binds to human chemokines with high affinity and with a specificity different from previously described for other gGs (Example 1).
  • the HSV-I and HSV-2 gG increase the ability of chemokines to induce cell migration of immune system, instead of inhibiting chemokine activity, either from T lymphocytes or monocytes (see Example 2).
  • gG increases the activity of chemokines by increasing intracellular signaling through the receptor, which is important because an alternative reason is that it increases the binding of chemokines to the cellular receptor, and is not the case (Example 1.5) .
  • results described in the present invention allow the possibility of using gG, preferably its soluble forms, of HSV (HSV-I and HSV-2) to be postulated as a pharmaceutical compound to treat processes or diseases that require cell migration of the immune system. It must be borne in mind that the migration of cells of the immune system and the infiltration of infected or damaged tissues plays an essential role in defense against pathogens and in inflammatory diseases.
  • HSV gG protein could be used to induce cell migration, preferably of cells of the immune system, mediated by chemokines by the therapeutic application of gG or its extracellular fragments already in its protein form. This could be achieved using the protein or peptide as a therapeutic active compound to be applied directly to a patient.
  • the potentiation of gG-mediated chemokine function can have a great effect on the pathogenesis of HSV due to the presence of a greater cell infiltrate and subsequent inflammation.
  • the design and generation of compounds that inhibit the function of gG allow its use as a therapy to treat infectious diseases caused by HSV, either HSV-I or HSV-2.
  • an object of the present invention is the use of a protein compound derived from the HSV glycoprotein (gG) inducer of chemokine activity, hereinafter use of a gG protein of the present invention, for the preparation of a medication or pharmaceutical composition for the treatment of a disease or disorder caused by a deficit of the Chemokine-mediated cell migration or that can be treated by using chemokines in vivo or ex vivo.
  • a protein compound derived from the HSV glycoprotein (gG) inducer of chemokine activity hereinafter use of a gG protein of the present invention, for the preparation of a medication or pharmaceutical composition for the treatment of a disease or disorder caused by a deficit of the Chemokine-mediated cell migration or that can be treated by using chemokines in vivo or ex vivo.
  • HSV glycoprotein-derived protein compound (gG) that induces chemokine activity refers to a protein molecule that mimics, increases the intensity or prolongs the duration of the inducing activity of the Cellular migration of a chemokine may be constituted by the complete sequence of the gG protein or a fragment thereof.
  • the chemokine system is complex, with more than 50 identified human chemokines classified into four classes (C, CC, CXC and CX3C) and about 20 receptors.
  • chemokine refers to a human chemokine, belonging to the illustrative title and without limiting the scope of the invention, to the following group: CCL22, CCL26, CCL25, CCL28, CXCL9, CXCLlO, CXCLIl , CXCL12- ⁇ , CXCL12- ⁇ , CXCL13, CXCL14 and XCLl.
  • HSV refers to a human herpes virus, either a herpes virus type 1 (HSV-I) or type 2 (HSV-2) (van Regenmortel, MHV, Fauquet, CM ., Bishop, DHL, Carsten, EB, Estes, MK et al., Virus taxonomy: Classification and nomenclature of viruses: Seventh report of the International Committee on Taxonomy of Viruses).
  • the term "disease caused by a deficit of migration mediated by chemokines or which can be treated by the use of chemokines in vivo or ex vivo” refers to a disease, disorder or pathology belonging, among others. by way of illustration and without limiting the scope of the invention, to the following group: ischemic cardiomeopathy, post-infarction therapy with spinal cord cells, acute coronary syndromes, carcinoma, bronchial-alveolar spontaneous carcinoma or chronic fever Q.
  • a particular embodiment of the invention is the use of the gG protein comprising the use of one or more amino acid sequences belonging to the following group: a) an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-I or HSV-2, b) an amino acid sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) an amino acid sequence that it comprises any sequence belonging to a), b) and c).
  • analogous is intended to include any amino acid sequence that can be isolated or constructed based on the sequence shown herein, for example, by introducing conservative or non-conservative amino acid substitutions. , including the insertion of one or more amino acids, the addition of one or more amino acids at any end of the molecule or the deletion of one or more amino acids at any end or within the sequence, and that mimics the inducing ability of the cell migration of a chemokine observed in the HSV gG protein in the present invention.
  • Analogous forms include the gG forms of the different strains HSV-I or HSV-2 existing in nature.
  • the gG of HSV-I and HSV-2 are expressed in the membrane of infected cells and also in the lipid envelope of the viral particle ( Figure IA). From the information described in the present invention ( Figure IB) and in the state of the art, a technician skilled in the art can isolate or construct a nucleotide sequence analogous to those described in the present invention for later use. .
  • an analogous amino acid sequence is substantially homologous to the amino acid sequence discussed above.
  • the term "substantially homologous” means that the amino acid sequences in question have a degree of identity of at least 30%, preferably of at least 85%, or more preferably of At least 95%.
  • Another particular embodiment of the present invention is the use of an activator compound of the invention where the amino acid sequence of c) is a fragment constituted by SEQ ID NO2, which represents the gG-ls form of HSV-I.
  • Another particular embodiment of the present invention is the use of an activator compound of the invention where the amino acid sequence of c) is a fragment constituted by SEQ ID N04, which represents for the gG-2s form of HSV-2.
  • Nucleotide sequences, fragments of the HSV-I and HSV-2 gG gene sequences, constructed in the present invention or similar fragments of these gG and which do not exist in nature can be used as molecular biology tools for expression of the corresponding proteins.
  • These "gG" nucleotide sequences, the genetic construct and the expression vectors containing them described in the invention can be isolated and obtained by an expert by employing techniques widely known in the state of the art (Sambrook et al. "Molecular cloning, a Laboratory Manual 2 nd ed., CoId Sping Harbor Laboratory Press, NY, 1989 vol 1-3).
  • another object of the present invention is a nucleotide sequence characterized in that it is an extracellular fragment of the HSV-I gG gene, preferably constituted by SEQ ID NO1, which codes for the HSV-I gG-ls form or by SEQ ID N03, which codes for the gG-2s form of HSV-2 (Example 1).
  • Said nucleotide sequences may be integrated into a gene expression vector that allows the regulation of recombinant expression thereof under conditions suitable for the production of the corresponding peptides or proteins that are a pharmaceutical active ingredient.
  • the use of said sequence fragments of HSV- and HSV-2 for the elaboration of biotechnological tools and to produce said proteins form part of the present invention.
  • an expression vector gG comprises, in addition to the nucleotide sequence gG or the gG genetic construct described in the invention, a promoter that directs its transcription (e.g., pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, signs of initiation and termination of transcription
  • a promoter that directs its transcription e.g., pT7, plac, ptrc, ptac, pBAD, ret, etc.
  • expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transiected or transformed with the gene or genes of interest.
  • the choice of the vector will depend on the host cell and the type of use to be performed. Therefore, according to a particular embodiment of the present invention said vector is a plasmid or a viral vector.
  • the obtaining of said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of microorganisms and eukaryotic cells different widely known methods can be used - chemical transformation, electroporation, microinjection, etc. - described in various manuals [Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2 nd ed. CoId Spring Harbor Laboratory, CoId Spring Harbor, NY].
  • lentivirus vectors include as an insert the melitin signal peptide followed by a histidine tag and a gG-ls fragment or gG-2s.
  • another object of the present invention is a peptide or protein derived or fragment of the HSV gG, either of the HSV-I or HSV-2 type, inducer of chemokine-mediated cell migration.
  • Another particular embodiment of the invention is an extracellular fragment protein of the HSV-I gG, preferably constituted by SEQ ID NO2, gG-ls protein form of HSV-I or SEQ ID NO4, gG-2s protein form. of HSV-2 (Example 1).
  • Another object of the present invention is a pharmaceutical composition or medicament for the treatment of diseases, disorders or pathologies caused or associated with a deficit of cell migration mediated by chemokines, hereinafter pharmaceutical composition of the present invention, which comprises a protein compound Chemokine-mediated cell migration inducer of the invention, in therapeutically effective amount together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.
  • compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
  • the term "therapeutically effective amount” refers to the amount of the agent or compound capable of developing axons of the neurons, calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the compounds, including age, patient's condition, severity of the alteration or disorder, and of the route and frequency of administration.
  • said therapeutic composition is prepared in the form of a solid form or aqueous suspension, in a pharmaceutically acceptable diluent.
  • the therapeutic composition provided by this invention can be administered by any appropriate route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.
  • the administration of the therapeutic composition provided by this invention is carried out parenterally, orally, intraperitoneally, subcutaneously, etc.
  • a particular embodiment of the invention is a pharmaceutical composition of the invention in which the gG protein or peptide belongs to the following group: a) an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-I or HSV-2, b) an amino acid sequence analogous to the sequence of a), c) a fragment of any one of the sequences of a) and b), and d) an amino acid sequence comprising any sequence belonging to a), b) and c).
  • Another particular embodiment of the present invention is the pharmaceutical composition of the invention in which the amino acid sequence is a gG protein whose amino acid sequence is constituted by the HSV-I gG protein.
  • Another particular embodiment of the present invention is the pharmaceutical composition of the invention in which the amino acid sequence is a gG protein whose amino acid sequence is constituted by the HSV-2 gG protein.
  • Another particular embodiment of the present invention is the pharmaceutical composition of the invention in which the amino acid sequence is a gG protein whose amino acid sequence of c) is a fragment consisting of SEQ ID NO2, which represents the gG-Is form. of HSV-I.
  • Another particular embodiment of the present invention is the pharmaceutical composition of the invention in which the amino acid sequence is a gG protein whose amino acid sequence of c) is a fragment consisting of SEQ ID NO4, which represents the gG-Is form. of HSV-2.
  • Another object of the invention is the use of the pharmaceutical composition of the invention, hereinafter use of the pharmaceutical composition of the invention, in a method of treatment or prophylaxis of a mammal, preferably a human being, affected by a disease, disorder or pathology with alterations in the chemokine-mediated cell migration or that can be treated by the use of chemokines in vivo or ex vivo consisting of the administration of said therapeutic composition in adequate dose that allows the recovery of the cell migration capacity, preferably of cells of the immune system.
  • Another particular object of the invention is the use of the pharmaceutical composition in which the disease with alterations in cell migration belongs, by way of illustration and without limiting the scope of the invention, to the following group: ischemic cardiomeopathy, post-therapy infarction with spinal cord cells, acute coronary syndromes, carcinoma, spontaneous bronchial-alveolar carcinoma or chronic fever Q.
  • the pharmaceutical composition of the present invention can be used in a treatment method in isolation or in conjunction with other pharmaceutical compounds.
  • Another object of the present invention is a method of identifying compounds useful for the treatment of infections caused by HSV characterized in that the HSV gG protein or an extracellular fragment thereof is used, either of the HSV-I type or HSV-I, as a therapeutic target.
  • Figure 1 (A) Representation showing the location of gG-1 and gG-2 in the virion and in the plasma membrane. GG-2 is also secreted to the extracellular medium after proteolytic processing. (B) Scheme of the constructions used in this invention. The protein domains of gG-1 and gG-2 and the regions of both cloned proteins are shown, including replacement of the Putative signal peptide of gG by melitin and the incorporation of histidine tag.
  • Figure 4 Chemical crosslinking assays showing the interaction between gG-ls and gG-2s with 125 I-CXCLlO (A) and 125 I-CCL25 (B).
  • the chemokine labeled with 125 I was incubated for 30 minutes with the gG. Subsequently, the BS3 crosslinker was added. The presence of a lower mobility complex than that of free chemokine in the lanes where samples containing gG were loaded indicates the interaction between gG and chemokine.
  • the arrows show the position of the gG / chemokine complex in the gel.
  • Figure 5 The gG present in the virion and in the infected cells binds chemokines.
  • A Chemical cross-linking assay with 125 I-CXCL12- ⁇ and viral particles obtained from cells infected with wild HSV-I (wt), HSV-1- ⁇ gG. The presence of a lower mobility complex is only observed when 125 I-CXCL12- ⁇ was incubated with wild HSV-I virions (wt) but not with those of HSV-1- ⁇ gG, demonstrating that the gG present in the Viral particle interacts with chemokines.
  • B Western blot showing the absence of gG expression in the HSV-1- ⁇ gG viral particles (top panel). Western blot showing that both wild HSV-I (wt) and HSV-1- ⁇ gG similarly express other viral proteins (lower panel).
  • C The gG present on the surface of infected cells binds chemokines. Graph showing the amount of 125 I-CXCLlO bound to uninfected cells or infected with wild HSV-I
  • HSV-1- ⁇ gG HSV-1- ⁇ gG
  • wild HSV-2 wt
  • Figure 6 The interaction between gG and chemokines takes place through the heparin binding site. Histogram showing the effect that heparin has on chemokine binding to gG. Increasing concentrations of heparin reduce the binding of CXCL12- ⁇ to gG-ls and of CXCL12-B to gG-2s.
  • Figure 7 The gG induces the migration of T cells (MOLT-4) and monocytes (MonoMac) mediated by chemokines.
  • MOLT-4 monocytes
  • MonoMac monocytes
  • A Increasing concentrations of gG-ls and gG-2s increase hCXCL12- ⁇ mediated migration, while gG-ls or gG-2s are not capable of inducing cell migration.
  • BGV-5 gG inhibits cell migration.
  • B The increase in chemokine-mediated cell migration is only seen when chemokine interacts with gG. 1:50 gG indicates a 1:50 molar ratio between chemokine and gG. 1: 100 gG indicates a 1: 100 molar ratio between chemokine and gG.
  • 1: 200 gG indicates a 1: 200 molar ratio between chemokine and gG.
  • Figure 8. The potentiation of the chemokine function mediated by gG is due to a greater activation of the chemokine receptor. Radioactively labeled GTPgamma incorporation assay (GTPgS) after receptor activation by SDFb chemokine with increasing amounts of gG. - • -, SDFb + 50X Gg-2s; -M-, SDFb; -A-, SDFb + lOOX Gg-2s; -x-, SDFb + 200X Gg-2s.
  • GTPgS GTPgamma incorporation assay
  • mice infected with the HSVdelgG virus showed fewer symptoms, especially between days 7 and 9 post-infection, than those infected with the HSV-lwt or HSV-lrevgG virus ( Figure 10c and 1Od).
  • HSV-I and HSV-2 are expressed in the membrane of infected cells and also in the lipid envelope of the viral particle ( Figure IA).
  • the membrane anchoring is due to the presence of a transmembrane region in gG.
  • gG is secreted to the extracellular medium after undergoing proteolytic processing.
  • gG-1 amino acids 28-142; gG-ls contains nucleotides 82-501 of gG-1 with accession number X14112; SEQ ID NO1 (nucleotides) and SEQ ID N2 (amino acids)) and of gG-2
  • amino acids 33-444 including the proteolytic processing site
  • gG-2s contains nucleotides 100-584 of gG-
  • SEQ ID NO4 amino acids
  • gG-ls and gG-2s respectively
  • Figure IB baculovirus expression vectors that allow the expression of proteins in insect cells
  • the signal peptide of the gG has been replaced by that of the melitin.
  • a 6x histidine tag has been incorporated at the N-terminal end, following the proteolytic cleavage site of the signal peptide (Figure IB).
  • Figure 2A shows purification by affinity chromatography of gG-ls (SEQ ID NO 2) and gG-2s (SEQ ID NO 4).
  • gG-ls and gG-2s were able to bind chemokines.
  • Chemokines were obtained from the Peprotech EC commercial house, London, United Kingdom.
  • gG-ls and gG-2s interact with human chemokines CCL22, CCL26, CCL25, CCL28, CXCL9, CXCLlO, CXCLIl, CXCL12- ⁇ , CXCL12- ⁇ , CXCL13, CXCL14 and CLl.
  • the binding affinities of some of these interactions are shown in Table 1. As can be seen, the affinity of the interaction is high, in the nanomolar range.
  • HSV-I and HSV-2 gGs were experimentally corroborated by conducting chemical cross-linking experiments, using chemokines marked with 125 I and BS3 as chemical crosslinker.
  • Chemokines marked with 125 I were obtained from the Amersham Biosciences commercial house.
  • the presence of a complex of lower electrophoretic mobility indicates the formation of a complex between gG-ls and gG-2s with 125 I-
  • gG present in the viral particle and in infected cells interacts with chemokines. Because the gG is in the lipid envelope of the HSV viral particle it was decided to check if the gG present in the virion was also able to bind chemokines. For this, wild HSV-I of strain SC16 was used and with HSV-I of the same strain in which the US4 gene, which codes for gG, has been interrupted by insertion of the lacZ gene (HSV-1- ⁇ gG ) (Balan P, Davis-Poynter N, Bell S, Atkinson H, Browne H, Minson T. (1994).
  • the purified virus was resuspended in Tris-HCl, pH 7.5; 0.15 M NaCl.
  • Chemical cross-linking experiments performed with purified virions and 125 I-CXCL12- ⁇ show the interaction between wild HSV-I and 125 I-CXCL12- ⁇ ( Figure 5A). This interaction is not observed with virions defective for gG-1 (HSV-I- ⁇ gG, Figure 5A).
  • the absence of gG-1 in the virions purified from supernatant from cells infected with HSV-1- ⁇ gG was demonstrated with the use of a monoclonal antibody that specifically recognizes gG-1 ( Figure 5B).
  • the cells were passed through a mixture of oil (1.5 parts of dibutyl thalate, Sigma D-2270; 1 part of bis (2ethylhexyl) thalate, Aldrich D20, 115-4) and washed with PBS to remove free chemokine not bound to cells (Dower SK, Kronheim SR, March CJ, Conlon PJ, Hopp TP, Gillis S, Urdal DL. (1985). Detection and characterization of high affinity plasma membrane receptors for human interleukin 1. J. Exp. Med. 162: 501-515). Subsequently, the number of accounts in each sample was determined with a gamma isotope counter.
  • 125 I-CXCLlO binds to cells infected with HSV-I more effectively than to uninfected cells or to cells infected with the HSV-1- ⁇ gG virus.
  • the binding of 125 I-CXCLlO to cells infected with HSV-2 was not as high as in the case of HSV-I. This may be because the chemokine binding domain of gG-2 is in the extracellular region, which is proteolytically processed and secreted to the medium, thereby reducing the amount of gG-2 on the cell surface.
  • GAGs glycosaminoglycans
  • GPCR protein-coupled transmembrane domains
  • chemokine domain responsible for the interaction with gG has been characterized.
  • the presence of increasing concentrations of a GAG such as heparin competes for the union of gG-1 ( Figure 6A) and gG-2 ( Figure 6B) with chemokines suggesting that both gG and heparin bind to chemokines by the same domain ( Figure 6).
  • GG and heparin do not interact (result not shown). Results have been obtained similar with CCL25, CCL28 and CXCLlO (result not shown).
  • 125 I-CXCL12- ⁇ to CXCR4 and 125 I-CCL25 to CCR9 binding assays were performed in the presence of gG-ls or gG-2.
  • the presence of increasing amounts of gG-ls or gG-2s did not affect the binding of chemokines to their receptor, while bovine herpesvirus gG 5 (BHV-5) 80% inhibited binding of 125 I-CXCL12- ⁇ to CXCR4 (result not shown).
  • the gG increases the activity of chemokines by increasing intracellular signaling through the receptor.
  • the binding of chemokine to its receptor triggers a signaling cascade that begins with the binding of
  • GTPgS to hCXCR4 receptor G protein expressed in A3.01 lymphocyte membranes, due to activation of the receptor by SDFb chemokine (stromal derived beta factor, CXCL12)
  • Figure 8 Pre-incubation of gG with chemokine increased the amount of GTP coupled to protein G, that is, increased receptor activation. This increase is best manifested with low concentrations of chemokine. Again, the presence of gG enhances the chemokine function reaching the maximum binding of GTP at a lower concentration of chemokine.
  • the potentiation of the chemokine function mediated by gG is due to a greater activation of the chemokine receptor.
  • MAPKs are involved in several cellular processes. In recent years it has been described that they play a important role in cell migration. Phosphorylation and, therefore, the activation of these proteins is necessary for cell migration directed towards a kinematic gradient (chemotaxis).
  • the addition of low concentrations of chemokine to the cells resulted in a mild phosphorylation of the Erkl / 2 and p38 MAPKs, detected in Western blot using a phosphorylated anti-Erkl / 2 antibody and phosphorylated anti-p38, respectively (Figure 9A).
  • Pre-incubation of chemokine with HSV gG increased this phosphorylation (Erkl / 2-Pi and p38-Pi panels).
  • Example 2. The HSV-I and HSV-2 gG induces the migration of chemokine-mediated T lymphocytes and monocytes.
  • Chemokines induce the migration of immune system cells.
  • vCKBP inhibit the Chemokine-mediated migration
  • vCKBP inhibits the Chemokine-mediated migration
  • Chem. 5: 833-848 In the case of non-human alpha herpesviruses, the presence of gG inhibits chemokine-mediated migration.
  • the present invention has investigated the ability of gG-ls and gGs-2 of HSV-I and HSV-2, respectively, to inhibit chemokine-mediated migration in in vitro migration assays using filters with a pore size of 3 ⁇ m The presence of chemokine at the bottom of the well induces the migration of cells through the pores. Prior incubation of HSV-I and HSV-2 gGs with chemokine does not inhibit CXCL12-B-mediated T-cell migration (MOLT-4).
  • FIG. 7A In contrast, the HSV-I and HSV-2 gGs induce chemokine-mediated migration ( Figure 7A). However, the gG of BHV-5 is able to inhibit this migration. HSG-I or HSV-2 gGs are not capable of inducing T-cell migration alone, without the presence of chemokine ( Figure 7A). The induction of chemokine-mediated migration is not dependent on cell type, as it is also appreciated when using mature monocytes ( Figure 7B). In addition, the interaction between the HSV-I or HSV-2 gGs and chemokine is necessary.
  • HSV-ldelgG gG deleted virus
  • HSV-lrevgG reverting virus
  • Viruses were purified using a sucrose gradient and titrated in VERO cells. The virus with gG deletion is attenuated since there was a lower mortality in C57BL6 mice infected either by the intranasal route with 10 x 10 7 plaque forming units (pfu) or by the intraperitoneal route with 10 x 10 4 pfu than in mice infected with the same pfu of the HSV-lwt or HSV-lrevgG virus ( Figure 10a and 10b).
  • mice infected with the HSVdelgG virus showed fewer symptoms, especially between days 7 and 9 post-infection, than those infected with the HSV-lwt or HSV-lrevgG virus ( Figure 10c and 1Od).

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Abstract

L'invention décrit l'utilisation d'un composé protéique dérivé de la glycoprotéine (gG) de HSV (HSV-1 y HSV-2), qui induit la migration cellulaire médiée par la chimiokine à des fins de préparation d'un médicament ou d'une composition pharmaceutique servant à traiter une maladie ou une pathologie résultant d'un déficit de la migration cellulaire, de préférence du système immunitaire. L'invention concerne également des compositions thérapeutiques qui comprennent lesdits composés et leurs applications thérapeutiques.
PCT/ES2007/070139 2006-07-20 2007-07-20 Glycoproteines de hsv ayant une activité inductrice de la migration cellulaire médiée par la chimiokine Ceased WO2008009777A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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US5656457A (en) * 1989-05-12 1997-08-12 Ciba Corning Diagnostics Corporation DNA sequence for the unique sequence herpes simplex virus type 2-glycoprotein G protein and method of expressing said unique sequence of HSV-2gG
WO2005092374A2 (fr) * 2004-03-22 2005-10-06 Istituto Superiore Di Sanita Virus d'herpes simplex recombinant et leurs utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5656457A (en) * 1989-05-12 1997-08-12 Ciba Corning Diagnostics Corporation DNA sequence for the unique sequence herpes simplex virus type 2-glycoprotein G protein and method of expressing said unique sequence of HSV-2gG
WO2005092374A2 (fr) * 2004-03-22 2005-10-06 Istituto Superiore Di Sanita Virus d'herpes simplex recombinant et leurs utilisations

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Title
BRYANT NEIL A. ET AL.: "Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins", THE EMBO JOURNAL ENGLAND, vol. 22, no. 4, 17 February 2003 (2003-02-17), pages 833 - 846 *
DATABASE GENBANK [online] Database accession no. (CS176891) *
DATABASE GENBANK [online] Database accession no. (I60459) *
DATABASE GENBANK [online] NORBERG P.R. ET AL.: "Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses", Database accession no. (AJ626502) *
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SEET B.T. ET AL.: "Viral chemokine-binding proteins", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 72, no. 1, 1 July 2002 (2002-07-01), pages 24 - 34 *
WEBB L.M. ET AL.: "Virally encoded chemokine binding proteins", MINI_REV. MED. CHEM., vol. 5, no. 9, 2005, pages 833 - 848 *

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