ES2317749B1 - USE OF A PROTEIC COMPOUND DERIVED FROM GLYCROPROTEIN (GG) OF HSV FOR THE PREPARATION OF A USEFUL PHARMACEUTICAL COMPOSITION FOR INDUCTION OF CELLULAR MIGRATION MEDIATED BY CHEMIOQUINS. - Google Patents

Abstract

Use of a protein compound derived from the HSV glycoprotein (gG) for the preparation of a composition Pharmaceutical useful for induction of mediated cell migration by chemokines.

The invention describes the use of a compound HSV glycoprotein (gG) derived protein (HSV-1 and HSV-2) inductor of the Chemokine-mediated cell migration for the development of a medicament or pharmaceutical composition for the treatment of a disease or disorder caused by a migration deficit cellular, preferably of the immune system. In addition, they are part of the invention therapeutic compositions comprising said compounds and their therapeutic applications.

Description

Use of a protein compound derived from the HSV glycoprotein (gG) for the preparation of a composition Pharmaceutical useful for induction of mediated cell migration by chemokines.

Technical sector

Biomedicine and biotechnology. Development of active therapeutic principles. Composition Development pharmaceuticals for the treatment of human diseases that they carry out alterations in the migration of system cells immune.

State of the art

During evolution, viruses have developed numerous strategies to modulate the immune system. Among them is to inhibit the function of chemokines. 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 Alon, R. ( 2001) Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines Nat. Immunol 2: 515-522; Proudfoot, AE, Handel, TM, Johnson, Z., Lau, EK, LiWang, P., Clark-Lewis, I., Borlat, F., Wells, TN, and Kosco-Vilbois, MH (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA . 100: 1885- 1890; Rot, A. (1992) Endothelial cell binding of NAP-1 / IL-8: role in neutrophil emigration. Immunol. Today , 13: 291-294). The chemokine system is complex, with more than 50 human chemokines identified classified into four classes (C, CC, CXC and CX3C) and about 20 receptors. 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. Immunol ., 3: 36-50; Alcami, A., Symons, JA, Collins, PD, Williams, TJ and Smith, GL (1998) Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus J. Immunol ., 160: 624-633; Lalani, AS, Barrett, JW and McFadden, G. (2000) Modulating chemokines: more lessons from viruses. Immunol. Today , 21: 100-106; Lalani, AS , Graham, K., Mossman, K., Rajarathnam, K., Clark-Lewis, I., Kelvin, D. and McFadden, G. (1997) The purified myxoma gamma virus interferon receptor homolog M-T7 interacts with the heparin -binding domains of chemokines. J. Virol . 71: 4356-4363; Murphy, PM (2001) Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immuno l ., 2: 116-122; van Berkel, V., Barrett, J., Tiffany, HL, Fremont, DH, Murphy, PM, McFadden, G., Speck, SH and Virgin, HI (2000) Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action . J. Virol ., 74: 6741-6747). Both poxviruses and herpesviruses code for secreted proteins with chemokine binding and inhibition capacity (viral chemokine-binding protein, vCKBP). The vCKBPs do not have sequence homology with the chemokine cell receptors or with each other, so each one represents a new structure capable of joining chemokines (Lalani, AS, Barrett, JW and McFadden, G. (2000) Modulating chemokines: more lessons from viruses. Immunol. Today , 21: 100-106; Murphy, PM (2001) Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immunol ., 2: 116-122).

The glycoprotein G (gG) present in some alpha herpesviruses has that capacity (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). Members of the 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.), Fields Virology. Lippincott Williams and Wilkins, Philadelphia, Vol. 2, pp. 2381-2397). This subfamily includes animal pathogens such as equine herpesviruses 1 and 3 (EHV-1 and EHV-3) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5) and human pathogens such as HSV-1 and HSV-2, which They replicate in oral and genital mucosa and varicella zoster virus (VZV). Some of the herpesvirus alpha glycoproteins present in the viral particle play a fundamental role in viral morphogenesis, cell binding and cell tropism. The role of glycoprotein G (gG), however, was unknown until it was discovered that gG encoded by alpha herpesviruses constitutes a new family of viral proteins with chemokine binding capacity (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). The gG of EHV-1, EHV-3, BHV-1 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. Because 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. However, no such activity was observed in the gG of HSV-1 and HSV-2 - which has very low sequence similarity with the gGs of equine and bovine viruses - with the chemokines used in this invention (CCL1, CCL2, CCL11 , CCL17, CCL20, CXCL1, CXCL10, CXCL21, CX3CL1 (see below in the present invention; Bryant et al ., 2003). In this regard, it has recently been shown that feline herpesvirus gG is capable of binding chemokines (B Costs, 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).

Both HSV-1 and HSV-2 are important human pathogens. HSV-1 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-1 and HSV-2) are pathogens humans with a high prevalence in the population, so that HSV-1 in adults is between 60 and 90% depending on the geographical area of the world. The reactivation of both viruses cause the production of viral particles with the appearance of lesions that have a great clinical impact. HSV-1 is the cause of corneal keratitis that It can cause blindness, meningitis and is the main viral cause of encephalitis due to its neurotropic character. So much HSV-1 as HSV-2 can cause encephalitis if the infection takes place in the nervous system central. Herpetic encephalitis is a focal brain process located in the frontotemporal and parietal areas of the brain. The Subacute encephalitis associated with HSV has been identified in 2% of the cases studied in patients with syndrome of the acquired immunodeficiency (AIDS). HSV poses a risk to infants since the infection during the third trimester of pregnancy and / or childbirth can lead to disseminated disease, failure of multiple organs, including the central nervous system, and death of the fetus or newborn. In the absence of an antiviral therapy Specific mortality exceeds 80% in disseminated disease and 50% in those patients who have symptoms only in the CNS. Most infected newborns develop Neurological disorders. A new aspect of HSV pathology which has acquired special relevance in recent years is the association of HSV infections with diseases neurodegeneratives like Alzheimer's. Unfortunately, the mechanisms employed by HSV-1 and HSV-2 in the modulation and evasion of the immune system, determinants of viral pathogenesis, are little known for what their compression may allow to develop new therapeutic approaches.

Description of the invention brief description

An object of the present invention constitutes it the use of a glycoprotein-derived protein compound (gG) HSV 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 migration deficit Chemokine-mediated cell.

A particular embodiment of the invention is constitutes the use of the gG protein where the use includes the use of one or more amino acid sequences belonging to following group:

to)

an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-1 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 any sequence belonging to a), b) and c).

Another object of the present invention is it constitutes a nucleotide sequence characterized because it is a extracellular fragment of the HSV-1 gG gene preferably constituted by SEQ ID NO1, which codes for the gG-1s form of the HSV-1 or by the SEQ ID NO3, which codes for the gG-2s form of the HSV-2 (Example 1).

Another object of the present invention is constitutes a peptide or derived protein or fragment of the gG of HSV, either HSV-1 or HSV-2, inducer of mediated cell migration by chemokines. Another particular embodiment of the invention is constitutes a gG extracellular fragment protein of HSV-1, preferably constituted by SEQ ID NO2, protein form gG-1s of HSV-1 or by SEQ ID NO4, protein form gG-2s of HSV-2 (Example 1).

Another object of the present invention is constitutes a pharmaceutical composition or medication 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 inducing the chemokine-mediated cell migration 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 constitutes a pharmaceutical composition of the invention in which The gG protein or peptide belongs to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-1 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 any sequence 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 being human, affected by a disease, disorder or pathology involved with alterations in chemokine-mediated cell migration consisting of the administration of said therapeutic composition in adequate dose that allows the recovery of the ability to cell migration, preferably from system cells immune.

Finally, another object of the present invention it is a compound identification procedure useful for the treatment of HSV infections characterized in that the HSV gG protein is used or a extracellular fragment thereof, whether of type HSV-1 or HSV-1, as target therapy.

Detailed description

The present invention is based on the fact that inventors have observed that the glycoprotein (gG) of viruses human HSV-1 and HSV-2, of great clinical relevance, joins human chemokines with high affinity, with a specificity different from that previously described for other gGs (Example 1). Also, contrary to what is described above in the case of gpes of herpesviruses alpha equines, bovines or felines, surprisingly, the gG of HSV-1 and HSV-2 increase capacity of chemokines inducing system cell migration immune, instead of inhibiting chemokine activity, since either from T lymphocytes or monocytes (see Example 2).

On the other hand, it has been shown that the ability of binding soluble gG (gGs) in cell membranes infected is greater with the gG of HSV-1 than with the HSV-2, indicating that the binding domain to Chemokines are found in the extracellular region of the gG, which it is proteolytically processed and secreted to the medium (example 1.3). In addition, it has been shown that the interaction between gG and chemokines take place through the domain of binding to chemokine glycosaminoglycans (Example 1.4).

On the other hand, it is likely that the ability to join chemokines and increase their function play a role fundamental in the viral cycle and in the pathogenesis mediated by HSV. This hypothesis is supported by two facts:

(I)

the chemokines to which gG binds are present in the tissues where the replicative cycle develops and HSV latency (mucous membranes and nervous system central),

(II)

the ability to bind chemokines that gG has present in the membrane of infected cells and in the virion (Example 1.3). This last data suggests that the interaction of gG with chemokines you can play an important role in initial stages of infection.

It should be noted that it is the first time that describes a secreted viral protein capable of activating the chemokine function. These results open the possibility of developing new therapeutic approaches for different Human diseases and pathologies. First, the results described in the present invention allow to postulate the possibility of using gG, preferably its soluble forms, of HSV (HSV-1 and HSV-2) as a compound pharmacist to treat processes or diseases that require Immune system cell migration. Must take into account that immune system cell migration and infiltration of infected or damaged tissues plays an essential role in the defense against pathogens and in inflammatory diseases.

It is therefore possible to think that one could use this same HSV gG protein to induce migration cell, preferably immune system cells, mediated by chemokines through the therapeutic application of gG or its Extracellular fragments already in their protein form. This is could get using the protein or peptide as a compound therapeutic asset to apply directly to a patient.

Second, the empowerment of the function of chemokines mediated by gG can have a great effect on the pathogenesis of HSV due to the presence of a greater infiltrate cellular and the consequent inflammation. The design and generation of compounds that inhibit the function of gG would allow its use as therapy to treat infectious diseases caused by HSV, either the HSV-1 or the HSV-2.

Therefore, 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 cell migration mediated by chemokines or that can be treated by the use of chemokines in vivo or ex vivo .

As used in the present invention the term "glycoprotein-derived protein compound (gG) of HSV chemokine activity inducer "refers to a protein molecule that mimics, increases intensity or prolongs the duration of the cell migration inducing activity of a chemokine may be constituted by the sequence complete with the gG protein or a fragment thereof. How has it commented above the chemokine system is complex, with more than 50 identified human chemokines classified in four classes (C, CC, CXC and CX3C) and about 20 receivers. In when the term "chemokine" refers to this patent, refers to a human chemokine, belonging to title illustrative and without limiting the scope of the invention, to Next group: CCL22, CCL26, CCL25, CCL28, CXCL9, CXCL10, CXCL11, CXCL12-?, CXCL12-?, CXCL13, CXCL14 and XCL1.

As used herein, the term "HSV" refers to a human herpes virus, either a herpes virus type 1 (HSV-1) 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).

As used in the present invention, 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, bronchio-alveolar spontaneous carcinoma or chronic fever Q.

A particular embodiment of the invention is constitutes the use of the gG protein that comprises the use of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-1 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 any sequence belonging to a), b) and c).

In the sense used in this description, the term "analogous" is intended to include any sequence of amino acids that can be isolated or constructed based on the sequence shown herein, for example, by the introduction of conservative amino acid substitutions or not conservatives, 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 in any end or inside the sequence, and that mimics the inducing ability of cell migration of a chemokine observed in the HSV gG protein in the present invention. The analogous forms include the gG forms of the different strains HSV-1 or HSV-2 existing in the nature.

The gG of HSV-1 and of HSV-2 (gG-1 and gG-2, respectively) are expressed in the membrane of the infected cells and also in the lipid envelope of the viral particle (Figure 1A). From the information described in the present invention (Figure 1B) and in the state of the art a Technical expert in the field of technology can insulate or build a nucleotide sequence analogous to those described herein  invention for later use.

In general, an analogous amino acid sequence is substantially homologous to the amino acid sequence commented above. In the sense used in this description, the expression "substantially homologous" means that the amino acid sequences in question have a degree of identity of at least 30%, preferably of at least one 85%, or more preferably, at least 95%.

A particular embodiment of the present invention is constituted by the use of a compound that induces invention in which the activating molecule is a gG protein whose amino acid sequence consists of the gG protein of HSV-1 (access number X14112; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide).

Another particular embodiment of the present invention is the use of an inducing compound of the invention in which the activating molecule is a gG protein whose amino acid sequence is constituted by the HSV-2 gG protein (accession number Z86099; http: //www.ncbi.nlm.nih.gov0/entrez/query. fcgi? CMD = search & DB = nucleotide).

Another particular embodiment of the present invention constitutes the use of an activating compound of the invention where the amino acid sequence of c) is a fragment constituted by SEQ ID NO2, which represents the form gG-1s of HSV-1.

Another particular embodiment of the present invention constitutes the use of an activating compound of the invention where the amino acid sequence of c) is a fragment constituted by SEQ ID NO4, which represents for the form gG-2s of HSV-2.

Nucleotide sequences, fragments of the sequences of the HSV-1 and HSV-2 gG genes, constructed in the present invention or similar fragments of these gGs that 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., Cold Sping Harbor Laboratory Press, NY, 1989 vol 1-3).

Thus, another object of the present invention is it constitutes a nucleotide sequence characterized because it is a extracellular fragment of the HSV-1 gG gene preferably constituted by SEQ ID NO1, which codes for the gG-1s form of the HSV-1 or by the SEQ ID NO3, which codes for the gG-2s form of the HSV-2 (Example 1).

Said nucleotide sequences may be integrated into a gene expression vector that allows regulation of recombinant expression thereof under conditions suitable for the production of peptides or proteins corresponding that are a pharmaceutical active ingredient. The use of said sequence fragments of HSV- and HSV-2 for the development of biotechnological tools and to produce said proteins form part of the present invention.

In general, a gG expression vector comprises, in addition to the gG nucleotide sequence 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, transcription initiation and termination signals ( tlt2 , etc.). ), polyadenylation signal, origin of replication, ribosome binding sequences (RBS), coding sequences of transcriptional regulators, (enhancers), transcriptional silencers (silencers), repressors, etc. Examples of appropriate 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 transfected 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. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. One strategy could be to use lentivirus to infect the target cells, as it is already being tried in other therapies (Ralph GS, Binley K, Wong LF, Azzouz M, Mazarakis ND (2006) Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors Clin Sci (Lond) 110: 37-46). An example of a particular embodiment is the expression vectors elaborated in the present invention: baculovirus vectors (pFastBac, Invitrogen) including as an insert the melitin signal peptide followed by a histidine tag and a gG-1s fragment or gG-2s.

Therefore, another object of the present invention it is a peptide or derived protein or fragment of gG HSV, either HSV-1 or HSV-2, inducer of mediated cell migration by chemokines. Another particular embodiment of the invention is constitutes a gG extracellular fragment protein of HSV-1, preferably constituted by SEQ ID NO2, protein form gG-1s of HSV-1 or by SEQ ID NO4, protein form gG-2s of HSV-2 (Example 1).

Another object of the present invention is constitutes a pharmaceutical composition or medication 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 inducing the chemokine-mediated cell migration of the invention, in therapeutically effective amount together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.

Pharmaceutical adjuvants and vehicles acceptable that can be used in said compositions are adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of compositions therapeutic

In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of agent or compound capable of developing axons of neurons, calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics own of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and of the route and frequency of administration

In another particular embodiment, 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 may 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. In one embodiment in particular, the administration of the therapeutic composition provided by this invention is carried out parenterally, by orally, intraperitoneally, subcutaneously, etc. A review of the different pharmaceutical forms of administration of medications and of the excipients necessary to obtain they can be found, for example, in the "Treaty of Farmacia Galenica ", C. Faulí i Trillo, 1993, Luzán 5, S.A. Editions, Madrid.

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A particular embodiment of the invention is constitutes a pharmaceutical composition of the invention in which The gG protein or peptide belongs to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of the HSV gG, either the gG form of HSV-1 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 any sequence belonging to a), b) and c).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is a gG protein whose amino acid sequence consists of the gG protein of HSV-1

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is a gG protein whose amino acid sequence consists of the gG protein of HSV-2

Another particular embodiment of the present invention constitutes 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 constituted by the SEQ ID NO2, which represents the gG-1s form of HSV-1

Another particular embodiment of the present invention constitutes 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 constituted by the SEQ ID NO4, which represents the gG-1s 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 that involves alterations in 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 cell migration capacity, preferably of immune system cells.

Another particular object of the invention is constitutes the use of the pharmaceutical composition in which the disease with alterations in cell migration belongs, to illustrative title and without limiting the scope of the invention, to next group: ischemic cardiomeopathy, therapy post-infarction with spinal cord cells, acute coronary syndromes, carcinoma, spontaneous carcinoma alveolar bronchus or chronic fever Q.

The pharmaceutical composition of the present invention can be used in a form treatment method isolated or in conjunction with other pharmaceutical compounds.

Finally, another object of the present invention it is a compound identification procedure useful for the treatment of HSV infections characterized in that the HSV gG protein is used or a extracellular fragment thereof, whether of type HSV-1 or HSV-1, as target therapy.

Description of the figures

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 the substitution of the putative signal peptide of gG with that of melitin and the incorporation of the histidine tag.

Figure 2: Expression and purification of gG-1s and gG-2s . (A) Purification of gG-1s and gG-2s by affinity chromatography. Fractions eluted with imidazole are shown. Protein detection was carried out by staining with Coomassie. (B) Detection of gG-1s and gG-2s purified by affinity chromatography by Western blot (WB).

Figure 3: Histogram showing chemokines that bind to gG-1s and gG-2s . The legend indicates the chemokine injected into the Biacore. The increase in relative units (RU) indicates the existence of an interaction between gG and injected chemokine.

Figure 4: Chemical crosslinking assays showing the interaction between gG-1s and gG-2s with125I-CXCL10 (A) and125I-CCL25 (B). The 125 I-labeled chemokine 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 with125I-CXCL12-? And viral particles obtained from cells infected with wild HSV-1 (wt), HSV-1- \ DeltagG. The presence of a lower mobility complex is only observed when the 125 I-CXCL12-? Was incubated with the wild HSV-1 (wt) virions but not with those of HSV-1- \ DeltagG, 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- \ DeltagG viral particles (upper panel). Western blot showing that both wild HSV-1 (wt) and HSV-1- \ DeltagG similarly express other viral proteins (lower panel). (C) The gG present on the surface of infected cells binds chemokines. Graph showing the amount of125I-CXCL10 bound to uninfected cells or infected with wild HSV-1 (wt), HSV-1- \ DeltagG and wild HSV-2 (wt). Cells infected with wild HSV-1 (wt) and wild HSV-2 (wt) bind more125I-CXCL10 than uninfected cells or those infected with HSV-1- \ DeltagG.

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-1s and of CXCL12-? To gG-2s.

Figure 7: The gG induces the migration of T cells (MOLT-4) and monocytes (MonoMac) mediated by chemokines . (A) Increasing concentrations of gG-1s and gG-2s increase hCXCL12-? Mediated migration, while gG-1s 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.

Examples of realization Example 1 G glycoproteins of HSV-1 and HSV-2 binds chemokines with high affinity 1.1.- Cloning and expression of G glycoproteins of HSV-1 and HSV-2

The gG of HSV-1 and of HSV-2 (gG-1 and gG-2, respectively) are expressed in the membrane of the infected cells and also in the lipid envelope of the viral particle (Figure 1A). The anchoring to the membrane is due to the presence of a transmembrane region in gG. In the case of HSV-2, in addition, gG is secreted to the medium extracellular after undergoing proteolytic processing. It has been cloned the soluble extracellular region of gG-1 (amino acids 28-142; gG-1s contains nucleotides 82-501 of gG-1 with access number X14112; SEQ ID NO1 (nucleotides) and SEQ ID N2 (amino acids)) and gG-2 (amino acids 33-444, including the processing site proteolytic; gG-2s contains the nucleotides 100-584 of gG-2 with number of access Z86099; SEQ ID NO3 (nucleotides) and SEQ ID NO4 (amino acids)) (gG-1s and gG-2s, respectively) in baculovirus expression vectors that allow expressing proteins in insect cells (Figure 1B). To improve the Protein secretion in insect cells has been replaced the signal peptide of gG with that of melitin. Further, to allow purification of proteins by chromatography of affinity, a 6x histidine tag has been incorporated at the end N-terminal, following the cutting site proteolytic signal peptide (Figure 1B). In Figure 2A, you can observe purification by affinity chromatography of the gG-1s (SEQ ID NO 2) and gG-2s (SEQ ID NO 4). Both gG-1s and gG-2s purified are recognized by a monoclonal antibody anti-histidines (Figure 2B). However, only the gG-1s is recognized by a monoclonal antibody anti-gG-1 (Figure 2B) (assigned by the Dr. Helena Browne, Cambridge, U.K.).

1.2.- The HSV-1 gGs and HSV-2 bind chemokines with high affinity

Both the gG-1s (SEQ ID NO 2) such as gG-2s (SEQ ID NO 4), purified by affinity chromatography, were covalently coupled to a Biacore CM5 chip in order to test the possible union of chemokines by the technique of Surface Plasmon Resonance (SPR). Subsequently, a binding test was performed with all the commercially available human chemokines to determine if the gG (gG-1s and gG-2s) was able to join chemokines. Chemokines were obtained from the house Commercial Peprotech EC, London, UK. As shown in the Figure 3A, gG-1s and gG-2s interact with human chemokines CCL22, CCL26, CCL25, CCL28, CXCL9, CXCL10, CXCL11, CXCL12-?, CXCL12-?, CXCL13, CXCL14 and CL1. 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.

TABLE 1 Chemokines used in Biacore to determine its possible interaction with gG-1s or gG-2s. The sign (+) indicates union. The sign (-) indicates lack of union. Affinity constants are shown (KD) for some of the interactions. Abbreviation: n.c: no calculated

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Subsequently, the interaction between the gGs of HSV-1 and HSV-2 and some Chemokines (CCL25 and CXCL10) were experimentally corroborated by conducting crosslinking experiments chemical, using chemokines labeled with125I and BS3 as chemical crosslinker Chemokines marked with125I They 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-1s and gG-2s with125I-CCL25 and 125 I-CXCL10 (Figure 4).

1.3.- The gG present in the viral particle and in cells infected 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-1 of strain SC16 was used and with HSV-1 of the same strain in which the US4 gene, which codes for gG, has been interrupted by insertion of the lacZ gene (HSV-1- \ DeltagG) (Balan P, Davis-Poynter N, Bell S, Atkinson H, Browne H, Minson T. (1994). An analysis of the in vitro and in vivo phenotypes of mutants of herpes simplex virus type 1 lacking glycoproteins gG, gE , gI or the putative gJ. J. Gen. Virol . 75 : 1245-1258). The supernatant was collected from uninfected BSC-1 cells or infected with wild HSV-1 or HSV-1-AgG and, after clarifying the supernatant by centrifugation at 1000 rpm for 10 minutes, the supernatant was passed through a 45% sucrose mattress prepared in 0.02 M Tris-HCl, pH 7.5; 0.15 M NaCl (22000 rpm, for 1 hour, SW28 rotor). 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-1 and 125 I-CXCL12-? (Figure 5A). This interaction is not observed with virions defective for gG-1 (HSV-1- \ DeltagG, Figure 5A). The absence of gG-1 in the virions purified from supernatant of cells infected with HSV-1-? Was demonstrated with the use of a monoclonal antibody that specifically recognizes gG-1 (Figure 5B). However, the expression of other viral proteins is observed in both wild HSV-1 and HSV-1-? When a polyclonal anti-HSV-1 antibody was used (Figure 5B).

The gG of HSV-1 and HSV-2 is also present in the membrane of infected cells but their function is unknown. Binding experiments of 125 I-labeled chemokines have been carried out to cells infected with HSV (wild HSV-1 (wt), HSV-1-? And HSV-2 wild). After 1 hour of incubation at 4 ° C, 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. As can be seen in Figure 5C, 125 I-CXCL10 binds to cells infected with HSV-1 more effectively than to uninfected cells or to cells infected with HSV-1- \ DeltagG virus . The binding of125I-CXCL10 to HSV-2 infected cells was not as high as in the case of HSV-1. 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. These experiments have also been carried out with125I-CXCL12-? Obtaining a similar result (data not shown).

1.4.- The interaction between the HSV-1 gG and HSV-2 with chemokines takes place through of the glycosaminoglycan binding domain present in the chemokines

The interaction between chemokines and glycosaminoglycans (GAGs) is necessary for their correct presentation to the receptors of 7 G-protein coupled transmembrane domains (GPCR) present in immune system cells and their function in vivo (Proudfoot, AE, Handel, TM, Johnson, Z., Lau, EK, LiWang, P., Clark-Lewis, I., Borlat, F., Wells, TN, and Kosco-Vilbois, MH (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA . 100: 1885-1890). The interaction of chemokine with the receptor triggers a cascade of intracellular signals that leads to cell migration. In the present invention the 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). Similar results have been obtained with CCL25, CCL28 and CXCL10 (result not shown). To determine whether gG interacts with chemokine through the receptor binding domain, 125 I-CXCL12-? To CXCR4 and 125 I-CCL25 to CCR9 binding assays were performed in the presence of gG -1s or gG-2. The presence of increasing amounts of gG-1s or gG-2s (up to 20 µg of protein) did not affect the binding of chemokines to their receptor, while bovine herpesvirus gG 5 (BHV-5) inhibited 80% the binding of125I-CXCL12-? to CXCR4 (result not shown).

Example 2 The gG of HSV-1 and HSV-2 induces Chemokine-mediated T lymphocyte and monocyte migration

Chemokines induce the migration of immune system cells. In all cases in which the capacity of the viral proteins that bind chemokines described to date to modulate chemokine activity has been investigated through the use of in vitro assays , vCKBP has been shown to inhibit chemokine-mediated migration ( Alejo A, Ruiz-Arguello MB, Ho Y, Smith VP, Saraiva M, Alcami A. (2006) A chemokine-binding domain in the tumor necrosis receptor factor from variola (smallpox) virus Proc. Natl. Acad. Sci. USA 103: 5995-6000; Webb LM, Alcami A. (2005). Virally encoded chemokine binding proteins. Mini. Rev. Med. 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-1s and gGs-2 of HSV-1 and HSV-2, respectively, to inhibit chemokine-mediated migration in in vitro migration assays using filters with a pore size of 3 \ mum. The presence of chemokine at the bottom of the well induces the migration of the cells through the pores. Prior incubation of HSV-1 and HSV-2 gGs with chemokine does not inhibit CXCL12-? Mediated T-cell migration (MOLT-4) (Figure 7A). In contrast, the HSV-1 and HSV-2 gGs induce chemokine-mediated migration (Figure 7A). However, the gG of BHV-5 is able to inhibit this migration. HSG-1 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 gGs of HSV-1 or HSV-2 and chemokine is necessary. Migration induction can only be seen when the chemokine that induces migration is one that interacts with gG (eg CXCL12-?) But not when gG is not able to bind it (eg CCL2) (Figure 7B ).

Claims (10)

1. Use of a protein compound derived from the HSV glycoprotein (gG) belonging to the following group: the HSV-1 gG-1s protein (sequence of amino acids SEQ ID NO2) and the gG-2s protein of HSV-2 (amino acid sequence SEQ ID NO4), for the development of a drug or pharmaceutical composition useful for induction of cell migration mediated by chemokines 2. Nucleotide sequence useful for the expression of a peptide characterized in that it is encoding an extracellular fragment of the HSV-1 gG protein constituted by SEQ ID NO1, which codes for the HSV-1 gG-1s form (SEQ ID NO2). 3. Nucleotide sequence useful for the expression of a peptide characterized in that it is encoding an extracellular fragment of the HSV-2 gG protein constituted by SEQ ID NO3, which codes for the HSV-2 gG-2s form (SEQ ID NO4). 4. Recombinant expression vector useful for the expression of a peptide characterized in that it comprises a nucleotide sequence according to claims 2 and 3. 5. Transformed or transfected cell useful for the production of a peptide characterized in that it comprises a nucleotide sequence according to claims 2 and 3 or a vector according to claim 4. 6. Protein derived or fragment of the HSV gG, either of the HSV-1 or HSV-2 type, useful as an inducer of chemokine-mediated cell migration characterized in that it belongs to the following group: SEQ ID NO2, gG-1s protein form of HSV-1, and SEQ ID NO4, protein form gG-2s of HSV-2. 7. Pharmaceutical composition or medicament useful for the induction of chemokine-mediated cell migration characterized in that it comprises an amino acid sequence belonging to the following group: amino acid sequence consisting of SEQ ID NO2, which codes for the gG-1s form of HSV-1 and amino acid sequence consisting of SEQ ID NO4, which codes for the gG-2s form of HSV-2, in therapeutically effective amount together with, optionally, one or more adjuvants and / or pharmaceutically acceptable carriers. 8. Use of the pharmaceutical composition according to claim 7 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 the chemokine-mediated cell migration consisting of administration of said therapeutic composition in adequate dose that allows the recovery of cell migration capacity, preferably of immune system cells. 9. Use of the pharmaceutical composition according to claim 8 characterized in that the disease with alterations in cell migration belongs to the following group: ischemic cardiomyopathy, post-infarction therapy with spinal cord cells, acute coronary syndromes, carcinoma, bronchial spontaneous carcinoma. alveolar or chronic fever Q. 10. Procedure for identifying compounds useful for the treatment of HSV-caused infections characterized in that a protein belonging to the following group is used: HSV-1 gG-1s protein (SEQ ID NO2) and HSV-2 gG-2s protein ( SEQ ID NO4).