WO1994028915A1

WO1994028915A1 - Hiv retrovirus infection inhibitor capable of interacting with cd26 receptor

Info

Publication number: WO1994028915A1
Application number: PCT/FR1994/000662
Authority: WO
Inventors: Ara Hovanessian; Christian Callebaut; Bernard Krust; Etienne Jacotot; Isabelle Marie
Original assignee: Institut Pasteur
Priority date: 1993-06-04
Filing date: 1994-06-03
Publication date: 1994-12-22
Also published as: AU1030195A; FR2707170A1

Abstract

Use of a compound to inhibit the entry of retroviral particles of an HIV-type human retrovirus, such as HIV-1 or HIV-2, in the target cells of an HIV retrovirus infected patient, which compound is capable of modifying the interaction between, on one hand, a CD26-type receptor present at the surface of the cells of a patient infected by said retrovirus, and on the other hand, the envelope glycoproteins of said HIV retrovirus. Recombinant cells capable of expreesing human CD4 and CD26 receptors are also provided.

Description

Inhibitors of HIV retrovirus infection, capable of interacting with the CD26 receptor.

The present application relates to means allowing the identification and the preparation of new inhibitors of infection by a human HIV retrovirus. It relates in particular to cells and recombinant organisms capable of expressing both the human CD4 receptor and the human CD26 receptor.

The human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS) and related disorders. So far two distinct but related types of retroviruses, the HIV-1 type and the HIV-2 type have been identified. Infection of CD4 + T lymphocytes, monocytes and macrophages by HIV requires the interaction of the virus env gene products with the cellular CD4 receptor (Klatzman D, et al. Nature 1984; 312: 767-768).

The HIV env gene codes for a precursor polyprotein which is cleaved by a cellular protease, leading to the production of extracellular or surface (SU) and transmembrane (TM) glycoproteins (Eiden LE, et al. Immunol. Today 1992; 13: 201-206). Surface protein, through non-covalent interactions with transmembrane protein, is exposed on the surface of infected cells or on the surface of viral particles.

The CD4 receptor is a glycoprotein composed of an amino-terminal extracellular region containing four domains related to the immunoglobulin family and of a carboxy-terminal region containing the transmembrane and cytoplasmic domains (Eiden et al). The first a terminal domain of the CD4 molecule appears to be the site principal binding of HIV surface proteins. Consequently, this binding induces a change in conformation of the SU-TM complex (complex formed by surface and transmembrane glycoproteins) allowing the interaction of the amino-terminal domain of the transmembrane protein with the cell membrane, thus causing the fusion of the membranes. viral and cellular (Marsh M, et al Immunol. Today 1988; 8: 369-371); Eiden LE et al. Immunol. Today 1992; 13: 201-206; Putney S. et al TIBS 1992; 17: 191-196). However before the fusion, the surface protein could undergo a modification by proteolytic cleavage, which in the case of the HIV-1 surface protein (GP120) seems to take place at the level of the third variable domain, commonly called "V3 loop", i.e., the primary neutralization domain (PND) (Moore JP, et al AIDS 1991; 5: S21-S33).

Various observations have so far suggested that the V3 loop plays a critical role in HIV-1 infection:

1) Neutralizing antibodies produced naturally in HIV-1 seropositive patients are mainly directed against the V3 loop (Goudsmit J, et al. Proc. Natl. Acad. Sci. USA 1988; 85: 4478-4482),

2) Monoclonal antibodies specific for the V3 loop do not affect the binding of gpl20 to the CD4 receptor but block the fusion process (Kinney-Thomas E, et al. AIDS 1988; 2: 25-29);

3) Mutations in the V3 loop generate virions with reduced infectivity (Grimaila RJ, et al. J. Virol. 1992; 66: 1875-1883);

4) Mutations in the V3 loop modify cellular tropism (Chesebro B, et al. J. Virol. 1992; 66: 6547-6554) and cause a modification in the phenotype of HIV as a virus inducing syncytium or not inducing syncytium (De Jong JJ, et al. J. Virol. 1992; 66: 6777-6780).

The V3 loop contains from 35 to 41, in particular approximately 36 amino acids whose cysteines at the two ends are connected, by a disulfide bridge forming a loop. The V3 loop contains conserved basic residues and in addition the sequence near the bound cysteines is similar in most isolates. (Clément GJ, et al. AIDS Res. Hum. Retroviruses 1991; 7: 3-16). Therefore, it appears that there is a selection pressure to keep the specific sequences in the V3 loop. Monoclonal antibodies directed against peptides (15-mer) representing the top ("crown") of the V3 loop have the capacity to neutralize HIV-1 (Langedijk JPM, et al. J. Gen. Virol. 1991; 72: 2519 -2526), this suggests that the residues (amino acids) at the top of the loop are involved in the functional interactions of the V3 loop.

So far no direct evidence has been established that cleavage of the V3 loop would be essential for HIV infection, although purified gpl20 could have been cleaved into 50 and 70 kDa fragments by different proteases: trombin and tryptase cleave the tryptic site (GPGR i AFVT), while cathepsin E cleaves a chymotrypsin-like site (GPGRAF i VT) (Clément GJ, et al. AIDS Res. Hum. Retroviruses 1991; 7: 3-16).

The inventors are interested in a particular sequence of the V3 loop of the surface glycoprotein of the envelope of the HIV-1 retrovirus, distinct from those which have been the subject of experience in the prior art, and in the corresponding sequences. HIV-2 and SIV retroviruses. They observed that a very large number of isolates (more than 90%) identified from HIV-1 have a peptide motif constituted by the amino acid sequence Gly-Pro-Gly (Glycine Proline - Glycine or according to the letter code GPG) and more particularly a preserved Gly-Pro motif. This GP or GPG motif also remains unchanged in vivo for several years in individuals infected with HIV-1, while other substitutions appear in the V3 loop (Holmback et al. J. Virol. 1993, 67: 1612-1619 ).

In addition, all of the identified isolates of HIV-1, HIV-2 and SIV present at the N-terminal end of the V3 loop a dipeptide motif conserved RP (Arginine - Proline). In addition, for HIV-2 and SIV, there is also a conserved dipeptide motif RP (Arginine-Proline), KP (Lysine-Proline), EP (Glutinic acid-Proline) or DP (Aspartic acid-Proline), at l C-terminal end of the V3 loop. Furthermore, for HIV 2 and SIV, there is also a conserved dipeptide motif RP (Arginine - Proline) or KP (Lysine - Proline) at the C-terminal end of the V3 loop.

Apart from the V3 loop, in the case of HIV-1, there are several conserved motifs, for example the sequences IPI (Isoleucine-Proline-Isoleucine), GPC (Glycine-Proline-Cysteine), PPG (Proline-Proline-Glycine ), located respectively at amino acids 185 and 208, of SU and 411 of TM, with respect to the consensus sequence of Myers et al (1992).

The inventors have investigated the extent to which these conserved peptide motifs could be associated with infection by the HIV retrovirus. They have demonstrated that these conserved motifs are capable of being the subject of an interaction with a molecule different from the CD4 receptor, present on cells infectable by the HIV retrovirus, or SIV.

The inventors have thus identified a new mechanism involved in infection by HIV or SIV retrovirus. In this regard, they have highlighted a new stage in the infection of lymphoid cells (including T cells) in patients infected with the HIV retrovirus. They have thus demonstrated that a step necessary for the entry of the retrovirus into cells (also called virus / cell fusion), involves a molecule different from the CD4 receptor. This step can also be involved in cell fusion (cell / cell fusion) which can occur after infection, to give rise to the formation of syncytia. According to this new mechanism, a receptor (DPPIV / CD26) present on the surface of cells susceptible to infection by the HIV or SIV retrovirus, is necessary for the infection of these cells by the retrovirus, in addition to the CD4 receptor already identified as a molecule required for infection of cells by the retrovirus.

In the case of infection of CD4 ^" cells, for example the neuronal cells SK-N-MC (Xi Ling Li et al J. Virol. 1990, 64: 1383-1387) or the epithelial cells (Fantini et al, PNAS , 1993, 90: 2700-2704) the inventors have identified the presence of this new receptor (DPPIV / CD26) which should intervene in the entry of the virus into CD4 cells ^" .

The inventors have now demonstrated that a surface cell protein interacts with the glycoproteins of the HIV or SIV envelope, in particular at the level of the V3 loop and is capable of cleaving it. This cleavage would take place at a site not identified as such until the present invention. The surface cellular protein is dipeptidyl peptidase IV (DPPIV) also called antigen or CD26 receptor (Barclay AN, et al. (1993). Facts book. London: Harcourt Brace Jovanich). DPPIV is a cell surface protease (EC 3.4.14.5) which generally has an exopeptidase activity and under certain conditions an endopeptidase activity (Nagatsu T, et al. Anal. Biochem. 1976; 74: 466-476 - Kudo M, et al J. Biochem. 1985; 97: 1211-1218 - Kenny AJ, et al. Biochem. J. 1976; 155: 169-182). Its enzymatic activity can be tested by the cleavage of synthetic dipeptides coupled to the chromogenic substrate p-nitroanilide (para-nitroanilide), these peptides having a sequence X-Pro-p-nitroanilide, to produce a peptide X-Pro and p- nitroaniline (Nagatsu T, et al. Anal. Biochem 1976; 74: 466-476) X which may preferably be an amino acid residue Glycine (Gly), Alanine (Ala), Lysine (Lys), Arginine (Arg), Glutamic Acid (Glu) and Aspartic Acid (Asp).

The CD26 antigen is widely expressed as a 110 to 120 kDa transmembrane glycoprotein, which is identical to the cell surface serine protease called dipeptidyl peptidase IV. This enzyme which is generally considered as an exopeptidase, cleaves dipeptides in the amino-terminal part of polypeptide substrates, since the penultimate residue is a proline residue; that is to say, this enzyme allows the cleavage of X-P-polypeptides, P being a Proline residue, when X is a free N-terminal amino acid (Kudo M, et al. J. Biochem. 1985; 97 : 1211-1218). Furthermore, DPPIV has been described as capable of catalyzing an "endopeptidase-like" cleavage, after a "GP-like" dipeptide motif within synthetic peptides (Kenny AJ, et al. Biochem. J. 1976; 155: 169 -182) and to bind to collagen (Beauvois B, et al. I. Marcel Dekker (Eds.) New York, 1991). DPPIV binds collagen but this binding does not affect its enzymatic activity (Hanski C, et al. Exp. Cell Res. 1988; 178: 64-72), thus suggesting the existence of attachment and catalytic sites in different DPPIV / CD26 domains. DPPIV is a glycoprotein comprising several oligosaccharide chains linked to N residues. The hydrophobic sequence near the amino-terminal end (signal peptide) of this ectoenzyme, functions as an anchoring domain to the membrane, the 6 amino acids N -terminals forming a cytoplasmic domain and the carboxy-terminal end of the protein constituting the extracellular domain (Ogata S, et al. J. Biol. Che. 1989; 264: 3596-3601 - Hong W, et al. J. Cell Biol. 1990; 111: 323-328). This enzyme exists in a wide variety of tissues including in lymphoid and non-lymphoid organs (Gorrel MD, et al. Cell. Immunol. 1991; 134: 205-215). It is expressed on the apical membranes of epithelial cells (brush borders and canalicular surfaces), endothelial cells, certain T lymphocytes, dendritic follicular cells and macrophages (Gorrel MD, et al Cell. Immunol. 1991; 134: 205-215 - McCaughan et al. 1990 Hepatology 11, 547-558 - Sannes PL, et al J. Histochem. Cytochem. 1983; 31: 684-690). The enzymatic activity was detected on different human cell lines of T and B origin, as well as on peripheral blood monocytes (Beauvois B, et al. I. Marcel Dekker (Eds.) New York, 1991). DPPIV / CD26 expression is weak in resting T cells, but increases considerably after activation; for this reason, DPPIV / CD26 is considered to be a T cell activation antigen.

The expressions DPPIV or CD26 will be used interchangeably in the rest of the text.

The identification of a new mechanism involving the CD26 receptor as mediator of infection in humans by an AIDS retrovirus, and more particularly its implication in the entry of the retrovirus into cells such as lymphocytes T, monocytes and macrophages, makes it possible to define new means for combating infection by an HIV or SIV retrovirus, and in particular gives access to new inhibitors of cell infection by the retrovirus.

The inventors have more particularly shown that DPPIV is a co-receptor for HIV, which is capable of binding the V3 loop, and / or other regions in the glycoproteins of the SU and TM viral envelope. This step concerning the interaction of the SU / TM complex with DPPIV / CD26 is essential for the penetration of HIV into CD4 T cells.

The subject of the invention is therefore compounds capable of modifying the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of HIV type -l or HIV-2 and on the other hand, the glycoproteins of the envelope of this HIV retrovirus.

It also relates to the compound CD 26, or a fragment of this compound, capable of associating with the glycoproteins of the envelope of HIV.

The term "HIV envelope glycoproteins" is not limited to the glycosylated form of the protein. On the contrary, it includes the interaction defined above with respect to the glycosylated form and / or the non-glycosylated forms of the envelope glycoproteins.

Similarly, the expression "the envelope glycoproteins" means that the interaction sought involves either a single one of these SU or TM glycoproteins, either these two glycoproteins individually or a complex formed by these glycoproteins, or their non-glycosylated forms. .

The invention also relates to the use of structural or functional inhibitors of the receptor CD26 to inhibit infection by an HIV or SIV retrovirus.

The invention also relates to the use of the compounds and inhibitors defined above, as active principle of pharmaceutical compositions. The compounds and inhibitors of the invention can be used to inhibit the entry of an HIV or SIV retrovirus into the target cells of an infected host and / or to inhibit the cell fusion of cells infected with an HIV or SIV retrovirus, with target cells of an infected host leading to the formation of syncytia and / or to inhibit cell death by apoptosis. Such compositions can in particular be used for the treatment of an infection with an HIV or SIV retrovirus. They can also be used for the preparation of vaccines.

Also within the scope of the invention, means for the screening of molecules capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus.

The invention therefore relates to the use of a compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus.

The compounds according to the invention have the property of inhibiting the entry of the HIV virus into the cells of the host and / or of inhibiting the formation of syncytia and / or the induction of apoptosis.

Inhibition of entry of the virus into the cells of a patient infected with an HIV retrovirus corresponds to an inhibition of the fusion of the cell membrane of the target cells of the host with the viral membrane.

The inhibition of the formation of syncytia corresponds to the inhibition of the fusion of cells infected with the HIV retrovirus, with target cells.

The invention also allows, according to a preferred embodiment, the inhibition of cell death by apoptosis.

The invention also relates to a pharmaceutical composition comprising, as active principle, the above compound.

The term “pharmaceutical composition” is intended to mean the combination of one or more compounds capable of having a treatment action vis-à-vis a pathogenic state or of preventing such a state, on the human body. or animal, on the other hand of constituents allowing the use, the administration and / or the conservation of this active ingredient in such a way that it can exert its action.

A compound having, according to the definition given above, the capacity to modify, that is to say in particular to alter or prevent the structural or functional interaction between the CD26 type receptor present on the surface of a patient's cells infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and, on the other hand, the glycoproteins of the envelope of this HIV retrovirus is generally a structural or functional inhibitor of the CD26 receptor.

By "inhibitor" of the CD26 receptor is meant a substance or a group of substances capable of altering or even preventing recognition by the CD26 receptor of the glycoproteins of the envelope of an HIV or SIV retrovirus, or of altering or prevent the binding of this receptor with this glycoprotein or alternatively altering or blocking the enzymatic function of the receptor with respect to peptide motifs of the X-Pro type, X being an amino acid residue in particular chosen from Gly, Ala, Lys residues , Arg, Glu, or Asp.

Such an inhibitor can directly block the CD26 receptor sites involved in the interaction with the glycoproteins of the HIV or SIV envelope and / or the site (s) giving rise to the enzymatic activity of the receptor (site catalytic), for example by settling on these sites. It can also act indirectly on these receptor functions, for example by steric inhibition of the sites in question.

The subject of the invention is therefore the use of a compound capable of modifying the interaction between the CD26 receptor and the glycoproteins of the envelope of the HIV retrovirus, the aforementioned compound having the property:

- to inhibit the recognition and / or the binding between the envelope glycoproteins and the CD26 receptor, and / or

- to inhibit the capacity of the CD26 receptor, to cleave a peptide bond after a motif of the X-P type in which X is a residue of an amino acid chosen from the amino acids Gly, Ala, Lys, Arg, Glu and Asp.

Are in particular usable as inhibitors of the virus / cell fusion and / or of the cell / cell fusion leading to the formation of syncytia, and / or cellular apoptosis, compounds capable of inhibiting the infection due to an HIV or SIV retrovirus, said compounds having the capacity to replace the envelope glycoproteins of an HIV or SIV retrovirus in interaction with the DPPIV / CD26 molecule, for example by constituting a substrate for the DPPIV enzymatic activity.

A specific inhibition of DPPIV activity on the cell surface leads to a decrease in the production of interleukin-2 (IL-2) and interferon-7 (IFN- ₇ ) by mononuclear cells stimulated by phorbol-ester and a reduced proliferative response to IL-2 by activated mitogenic T lymphocytes (Schôn E, et al. Scand. J. Immunol. 1989; 29: 127-132). In view of these observations, it has been suggested in the prior art that DPPIV could be involved in the induction and activation of cytokines which control lymphocyte proliferation (Beauvois et al; Schόn).

DPPIV is present on both quiescent CD4 ⁺ and CD8 ⁺ T cells and its expression is increased by activation of T cells (Gorrel MD, et al. Cell. Immunol. 1991; 134: 205-215). Research on human T lymphocytes has shown that the expression of DPPIV is associated with that of CDw29 ⁺ , marker of memory cells and with the activation induced by stimulation of CD2 or CD3. These functions are confirmed by the ability of anti-DPPIV antibodies to modulate surface expression of DPPIV. These antibodies also promote the increase in the proliferative response of T cells treated with anti-CD3 or anti-CD2 antibodies, a response which is preceded by an increase in the mobilization of Ca ²⁺ ions. DPPIV is physically associated with a known membrane-bound CD45 protein, such as tyrosine phosphatase. (Torimoto Y, et al. J. Immunol. 1991; 147: 2514-2517) (Alexander D, et al. Immunol. Today 1992; 13: 477-481).

These observations suggest that DPPIV plays a role in regulating the activation of human T cells through its interaction with CD45, itself known to intervene in lymphocyte activation.

The interaction of the envelope glycoproteins of HIV or SIV retroviruses with DPPIV / CD26, could modify the balance of the signals transduced during Lymphocyte activation and causing cell death by apoptosis (Callebaut C. and Jacotot E., 1992; Master thesis, University of Paris 11). Indeed, the interaction of the envelope retrovirus glycoproteins with the lymphocytes initiates a series of events leading to apoptosis.

The capacity of the compound which can be used in the context of the invention, to modify the interaction of the CD26 receptor with respect to the glycoproteins of the envelope of the HIV retrovirus, can be determined by the test comprising the following steps:

the bringing into contact on the one hand of cells comprising on their surface the CD26 receptor, on the other hand of an amount equal to TCID50, of a human HIV retrovirus, in particular HIV-1 or HIV-2, or d '' a SIV simian retrovirus,

- the incubation of the above cells and of the HIV or SIV retrovirus at 37 ° C. for a time sufficient to allow the penetration of the retrovirus into the cells, in the presence of a determined quantity of the test compound,

washing the cells to eliminate the retroviruses absorbed on the cells, eliminating the extracellular virus and quantifying the intracellular virus, centrifuging to separate the retroviral proteins, for example at 1000 g, and recovering the supernatant,

- detection of HIV proteins.

According to a particular embodiment of the invention, the step of bringing the test defined above into contact with a human HIV retrovirus, in particular HIV-1 or HIV-2 or a simian retrovirus SIV, in a amount equal to TCID 50, is carried out with cells comprising on their surface, CD4 receptors and CD26 receptors. Particularly interestingly, these receptors are species specific and preferably are the human CD4 and CD26 receptors.

According to another particular embodiment of the invention, the capacity of a suitable compound for the implementation of the invention, to modify the interaction of the CD26 receptor with respect to the glycoproteins of the envelope of the retrovirus HIV, can be determined by the test comprising the following steps:

1- bringing into contact, on the one hand, cells comprising on their surface the CD26 and / or CD4 receptors, on the other hand an amount equal to TCID50, of a human HIV retrovirus, in particular HIV-1 or HIV -2, or a SIV simian retrovirus,

2- the incubation of the above cells and of the HIV or SIV retrovirus at 37 ° C. for a time sufficient to allow penetration of the retrovirus into the cells, in the presence of a determined quantity of the compound tested,

3- washing the cells to eliminate the retroviruses absorbed on the cells,

4- the treatment of cells to eliminate the remaining extracellular retroviruses, for example by controlled digestion with trypsin,

5- the preparation of cytoplasmic extracts by treatment of the cells with an extraction buffer, for example with a buffer containing 20 M Tris-HCl, at pH 7.6, 0.15 M NaCl, 5 mM MgCl ₂ , 0.2 mM PMSF, 100 units / ml aprotinin and 0.5% Triton X-100,

6- centrifugation to separate the retroviral proteins, for example at 1000 g, and recovery of the supernatant,

7- the detection and if necessary the measurement of the protein concentration of the HIV or SIV retrovirus, for example core protein, obtained in the supernatant, for example by an ELISA test.

An alternative to this test is to culture the cells after the digestion step controlled by trypsin and to test the viral production after 2 or 3 days. Thus, after step 3, the cells can be incubated in the culture medium and the viral production measured, for example after 3 or 4 days.

Preferably, the cells used for carrying out these tests express species-specific CD4 and CD26 receptors, in particular human molecules, when it is a question of testing the interaction of the CD26 receptor with an HIV retrovirus. .

According to a preferred embodiment of the invention, the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, is a peptide or non-peptide inhibitor.

The compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is for example an antibody directed against the CD26 receptor or an epitope which does not affect the catalytic activity of the DPPIV enzyme.

Such an antibody is, for example, capable of binding to an epitope of the CD26 receptor in such a way that the interaction with the envelope glycoproteins of HIV or SIV is inhibited either directly, or indirectly for example by steric hindrance.

According to another embodiment of the invention, the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a monoclonal antibody directed against an epitope of the CD26 receptor comprising all or part of the G SYG sequence (Glycine-Tryptophan -Serine-Tyrosine-Glycine) which is the potential catalytic site of CD26 (Tanaka T et al J. Immunol., 1992, 149: 481-486).

The inventors have demonstrated that the catalytic site of CD26 can involve three amino acid sequences of the amino acid sequence of the CD26 molecule, one being the GWSYG chain, the others being the HGT and chain respectively. NNDI. Within these sequences, the inventors have shown the particular interest of the residues S (serine), H (histidine) and D (aspartic acid).

An antibody capable of being used for carrying out the invention is advantageously such that the complex which it forms with the CD26 receptor alters or neutralizes the catalytic activity of the CD26 receptor.

As an example of another type of compound of the invention, there may be mentioned the antibodies directed against an epitope of the V3 loop of an HIV or SIV retrovirus containing the Gly-Pro (GP) motif or a GP- motif like such as an Ala-Pro (AP), Lys-Pro (KP), Arg-Pro (RP), Glu-Pro (EP) or Asp-Pro (DP) motif.

According to another embodiment of the invention, the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is the tripeptide IPI or the tripeptide KPR or a peptide having in its structure, the conformation of the tripeptide IPI or of the tripeptide KPR and in particular having in its structure a peptide or a polypeptide containing a sequence carrying the inhibitory function of the peptide IPI and possibly having a symmetry at the level of its amino acid sequence, the center of symmetry of this sequence preferably consisting of a native or modified Proline residue.

According to another embodiment of the invention, a compound of interest contains a recognition motif contained in a substrate of the DPPIV enzyme, or a motif having a conformation sufficiently close to that of the above recognition motif, to be recognized by the DPPIV enzyme.

Such a compound is capable of behaving like a substrate of the DPPIV enzyme and therefore of occupying the enzymatic site of the molecule, preventing the binding between the DPPIV molecule and the envelope glycoproteins of HIV or SIV.

This compound capable of replacing the enzymatic substrate of DPPIV is of peptide or non-peptide nature. It may, for example, be a molecule having the three-dimensional conformation of the recognition motif normally active at the level of the DPPIV substrate.

In the remainder of the text, the expression “recognition motif contained in a substrate of the DPPIV enzyme” will sometimes be replaced by the expression “recognition motif of the substrate of the DPPIV enzyme”.

Certain compounds meeting this definition may also be suitable for inducing production of neutralizing antibodies against HIV infection or SIV.

A compound of interest comprising a motif for recognizing the substrate of the DPPIV molecule contains, for example, a peptide motif chosen from the sequences GP, RP, KP DP or EP, or a motif chosen from the sequences GPG, RPG, KPG DPG or EPG.

According to a first embodiment of the invention, this motif of enzyme substrate recognition is presented in a structural context analogous to the peptide environment existing at the level of the V3 loop of the glycoprotein of the outer envelope of HIV or SIV. In this regard, it should be noted for example that the presence of the glycine residue after the proline residue contributes to the formation of a β-elbow type structure favorable to the exposure of proline. According to another variant, the above motif is in a peptide environment different from that existing at the level of the V3 loop.

Compounds containing the substrate recognition motif are, for example, the peptides corresponding to one of the sequences GPGRAF, KRPGNK, RPGNK, KRPRQ or EPGKN.

These peptides can be isolated from HIV or SIV antigens or chemically synthesized. The amino acid residues they contain can be levorotatory or destrogyrous. Advantageously, all the residues of the same peptide are of the levorotatory type or all the residues of the same peptide are of the dextrorotatory type.

Such a compound retains, if necessary, the ability to be cleaved by the enzymatic action of DPPIV.

According to another embodiment of the invention, a pharmaceutical composition is characterized in that the recognition motif is modified so as to prevent the enzymatic cleavage activity normally exerted by the DPPIV on this motif.

An appropriate modification can be carried out at the level of the peptide bond following and / or preceding the Proline residue of the recognition motif, in order to prevent the cleavage of this bond and therefore in order to decrease or suppress the sensitivity to peptidases.

The usual techniques for modifying peptide bonds can be used.

Mention will be made in particular without limitation of the modifications of the peptide bond -CONH- leading to the formation of a reduced bond (CH NH), retro inverso (NHCO), methylene-oxy (CH ₂ -0), thiometylene (CH ₂ - S), carba (CH ₂ CH ₂ ), ketomethylene (CO-CH ₂ ), hydroxyethylene (CHOH-CH ₂ ), Aza (NN), E-alkene, -CH = CH- or carba -CH ₂ -CH ₂ .

The subject of the invention is also the nucleotide sequences coding for the compounds containing the motif for recognition of the substrate of the enzyme DPPIV. These sequences can optionally be administered to a patient for the purpose of treating or preventing AIDS; With regard to administration techniques, reference is made to the publication by Wang et al (PNAS USA, vol 90 pp 4156-4160, May 1993) or to the international patent application published under the number WO 90/11092, the October 4, 1990.

These compounds described above for their ability to inhibit virus / target cell and / or cell / cell fusion and / or cell apoptosis and thus to constitute active principles of pharmaceutical compositions, and in particular compounds containing a motif of the type GP, RP, KP, EP, DP or a tripeptide motif such as GPG, RPG, KPG, EPG, DPG, have the remarkable property of being able to enter into the composition of vaccines against infection with an HIV or SIV retrovirus although the sequences of the recognition motif are not necessarily accessible on the gpl20 glycoprotein. Their activity and in particular their capacity to induce antibodies against the envelope glycoproteins can indeed manifest themselves whatever the isolate of the HIV retrovirus (of the HIVl or HIV2 type in particular) or SIV in question, since this activity is defined from of sequences conserved between different isolates of the HIV retrovirus sequences being further conserved over time in a patient infected with the virus.

If necessary, the compound which can be used as active principle will be combined with a substance protecting it in vivo against the action of cellular proteases, or in general with any carrier molecule or system designated by the term complex, promoting its properties for example of recognition of its target or its antigenic or immunogenic properties.

Thus, the compound which can be used for carrying out the invention and which can enter into the constitution of the pharmaceutical composition is advantageously accompanied or contained in a heterologous structure, or polymerized so as to promote its activity of inhibition or prevention of 1 ' HIV or SIV retrovirus infection in vivo

By way of example, this compound can be included within a synthetic matrix of peptide type, in particular to form a structure of MAP type. Such structures have been described as well as their mode of preparation in the publication by Tarn, JP (PNAS, USA, vol. 85, 5409-5413, August 1988). The inclusion of the active principle of the drug within MAP-type structures has the advantage of promoting its inhibitory properties with respect to infection by an HIV or SIV retrovirus. According to another embodiment of the invention, the compound described above, when it is of peptide nature, is produced in the form of a recombinant molecule, for example in the form of a fusion protein, in particular with a HBsAg type antigen, for example by insertion into the S or pre-S sequence of the antigen. Techniques for producing fusion proteins are well known to those skilled in the art and reference will be made, for example, to the publication by Delperoux et al (Science, vol. 233, pp. 472-475, July 25, 1986).

According to another embodiment of the invention, the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a soluble form of the CD26 receptor, in particular the CD26 receptor lacking its transmembrane sequence.

The invention also relates to a method for the in vitro screening of compounds capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of the type HIV, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, characterized in that it comprises the test comprising the following steps:

- incubation of the above cells and of the HIV or SIV retrovirus at 37 ° C. for a sufficient time to allow the retrovirus to penetrate into the cells, in the presence of a determined quantity of the test compound,

- detection of HIV proteins.

According to a first embodiment of this method, the screening is carried out after having brought the HIV or SIV retrovirus into contact, with cells comprising on their surface, the CD4 receptor and the CD26 receptor and preferably specific CD4 and CD26 receptors species, including human receptors.

Preferably, this process comprises the following stages:

the bringing into contact on the one hand of cells comprising on their surface the CD26 and CD4 receptors, on the other hand of an amount equal to TCID50, of a human HIV retrovirus, in particular HIV-1 or HIV-2, or a simian SIV retrovirus,

- the incubation of the above cells and of the HIV or SIV retrovirus at 37 "C for a time sufficient to allow penetration of the retrovirus into the cells, in the presence of a determined quantity of the test compound,

washing the cells to eliminate the retroviruses absorbed on the cells, treating the cells to eliminate the remaining extracellular retroviruses, for example by controlled digestion with trypsin, preparing the cytoplasmic extracts by treating the cells with an extraction buffer, for example with a buffer containing 20 mM Tris-HCl, at pH 7.6, 0.15 M NaCl, 5 mM MgCl ₂ , 0.2 mM PMSF , 100 units / ml aprotinin and 0.5% Triton X-100, centrifugation to separate the retroviral proteins, for example at 1000 g, and recovery of the supernatant,

the detection and, where appropriate, the measurement of the protein concentration of the core of the HIV or SIV retrovirus, obtained in the supernatant, for example by an ELISA test.

An alternative to this test is to culture the cells after the digestion step controlled by trypsin and to test the viral production after 2 or 3 days.

Also within the scope of the invention are the compounds capable of inhibiting in vivo infection by a human HIV retrovirus, in particular a retrovirus of the HIV-1 or HIV-2 type, capable of modifying or inhibiting the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with an HIV retrovirus and, on the other hand, the glycoproteins of the HIV envelope, said compound being of peptide or non-peptide nature .

Particularly advantageous compounds are for example:

- an antibody directed against the CD26 receptor, in particular a monoclonal antibody directed against an epitope comprising all or part of the GWSYG sequence;

- an antibody directed against an epitope contained in the site formed by the sequences GWSYG, HGT, NNDI of the molecule CD26;

- an antibody directed against an epitope of the V3 loop of an HIV or SIV retrovirus containing the Gly-Pro (GP) motif or a GP-like motif such as an Ala- motif Pro (AP), Lys-Pro (KP), Arg-Pro (RP), Glu-Pro (EP) or Asp-Pro (DP) or a tripeptide motif of GPG, APG, KPG, RPG, EPG or DPG type; an antibody having these properties, can be an antibody directed against one of the sequences given in Table 9,

- A peptide or a polypeptide having in its structure, the conformation of the tripeptide IPI and in particular a peptide or a polypeptide having a symmetry at the level of its amino acid sequence, the center of symmetry of this sequence preferably being constituted by a residue Native or modified Proline,

a peptide, a polypeptide with the exception of the unmodified KPR or RPGFSPFR peptides, comprising a recognition motif contained in the substrate of the DPPIV enzyme, for example a GP, KP, RP, EP or DP motif or a tripeptide motif of type GPG, KPG, RPG, EPG, or DPG, or a structural and / or functional analogue of this peptide or of this polypeptide such as a non-peptide molecule reproducing the essential three-dimensional conformation of this peptide or of this polypeptide as it exists in the V3 loop external glycoproteins of the HIV or SIV envelope or, where appropriate, in another peptide environment; by way of example, the peptides GPGRAF, KRPGNK, RPGNK, KRPRQ or EPGKN will be mentioned;

a fragment of the CD26 molecule, in particular its soluble form, for example a fragment containing all or part of the GWSYG, HGT and NNDI sequences, or a fragment containing the site of interaction with the outer envelope glycoproteins of HIV or SIV;

- A nucleotide sequence encoding a motif for the recognition of the enzymatic substrate of the DPP IV molecule.

These compounds can be coupled to a carrier molecule or be associated with or integrated into a system, peptide or not, having the capacity to protect them against the action of proteases in vivo.

When it is a question of peptides or peptide derivatives, they are advantageously modified to decrease, even eliminate their sensitivity to proteases in vivo. Such modifications have been described in the preceding pages. The above compounds can also be combined with substances such as interleukin, with adjuvants such as Poly (A) -poly- (U) described by Hovanessian A et al (Immunology Today, vol 9, No. 6, 1988, pp. 161-162) and Krust et al.

In particular, the subject of the invention is the use of a 1F7 monoclonal antibody or of the IPI peptide or of an inhibitor having the same inhibitory function with respect to HIV, of peptide or non-peptide nature, for the treatment of cells likely to be infected with HIV or SIV.

The invention also relates to compositions containing several inhibitors of an infection by an HIV or SIV retrovirus. Such compositions may contain a mixture of the compounds according to the invention with anti-virals such as AZT, DDI, DDC or with antibodies, in particular anti-peptidase or anti-protease or any antibody having neutralization properties vis -in relation to HIV or SIV.

By way of example, also falling within the scope of the invention, compositions containing several types of inhibitors of the interaction between CD26 and the glycoproteins of the outer envelope of HIV or SIV, chosen from those which have been described below -above.

Such a composition can contain an anti-CD4 antibody and a CD26 receptor inhibitor, as described above. It can also be a composition containing an antibody having neutralization properties with respect to HIV (anti-HIV antibody) or SIV and an anti-CD26 antibody or a CD26 receptor inhibitor.

According to a particular aspect of the implementation of the invention, the active compounds or principles can be administered to the patient so as to be released with delay in the organism; thus they can be combined with calcium phosphate or aluminum hydroxide.

Can still be used, to prepare inhibitors of the interaction between CD26 / DPP IV and the envelope glycoproteins of HIV or SIV, molecules defined from the substrate of the DPP IV molecule as described by Fischer G et al (Pharmazie 38, 1983), Heinz J et al (biochemica and Biophysica Acta 954 (1988) 161-169), Brandt W et al (J. of Computer Aidée Molecular Design 6, 1992, 159-174).

The invention also relates to the use of the polypeptide sequences of Table 9, for preparing antibodies having the capacity to inhibit the interaction between on the one hand a CD26 type receptor present on the surface of cells of a patient infected with a human HIV retrovirus, in particular of the HIV-1 or HIV-2 type, and on the other hand the envelope glycoproteins of this retrovirus.

The invention further relates to a polyclonal or monoclonal antibody directed against an antigenic sequence of HIV or SIV, capable of modifying, in particular to inhibit the interaction between on the one hand, a receptor of the CD26 type present on the surface of cells. of a patient infected with a human HIV retrovirus, in particular of the HIV-1 or HIV-2 type, and on the other hand the envelope glycoproteins of this retrovirus.

The invention also relates to pharmaceutical compositions or compounds among those described above, for preventing or treating cellular apoptosis. Also within the scope of the invention are sequences of the V3 region of the HIV envelope glycoproteins, characterized in that they have the capacity to induce, for example idiotypic antibodies capable of interacting with the CD26 receptor.

The invention also relates to a recombinant cell, characterized in that it is capable of jointly expressing human CD4 and CD26 receptors.

Recombinant cells of interest are advantageously eukaryotic cells and preferably animal or human cells, for example human HeLa cells or murine cells such as NIH3T3 cells.

These cells can be used for the in vitro screening of substances capable of behaving as inhibitors of the interaction between the CD26 receptor and the envelope glycoproteins of an HIV or SIV retrovirus, for example in the context of tests which have been described above.

The invention also relates to a transgenic animal characterized in that its somatic cells and / or its germ cells comprise a sequence coding for the human CD4 receptor and a sequence coding for the human CD26 receptor, under conditions allowing the joint expression of these two receivers.

As regards the transgenic animal, the invention excludes man.

Transformation of the animal's somatic cells alone makes it possible to obtain expression of the CD4 and CD26 receptors in this animal but excludes transmission to their descendants. On the contrary, the modification of the germ cells allows the transmission of genes or coding sequences of interest, in subsequent generations of the animal.

According to a particular embodiment, the transgenic animal can be obtained by introduction into the cells of the animal at a stage of early embryonic development, for example not exceeding 8 cells, of a sequence coding for the human CD4 receptor and of a sequence encoding the human CD26 receptor, under conditions allowing the joint expression of these two receptors.

The modification of the cells by the coding sequence or the gene coding for the CD4 and CD26 receptors is advantageously carried out in the first stages of embryonic development and this in order to ensure in the developed animal the transformation of the whole or almost all of these cells.

Different techniques are available for inserting a gene or a sequence into the cells of an animal embryo. We cite in particular the publication by Brinster RL et al, PNAS, USA, vol. 82, pages 4438-4442, July 1985.

An animal of interest in the context of the invention may be the animal resulting from the development of the embryo which has been transformed to introduce the genes or the sequences of interest therein. It may also be a descendant of such an animal.

Transgenic animals of interest in the context of the invention are for example mammals and in particular rodents such as mice. We can also use monkeys.

The subject of the invention is also a method for obtaining a non-human transgenic animal comprising in its somatic cells and / or in its germ cells a sequence coding for the human CD4 receptor and a sequence coding for the human CD26 receptor, in conditions allowing the joint expression of these two receptors comprising the steps of:

- the introduction of a sequence encoding the human CD4 receptor, for example a genomic sequence or a cDNA sequence, into the cells of the animal, at an early stage of embryonic development, for example not exceeding 8 cells ,

The introduction into an animal ature of the same species of a sequence coding for the twin CD26 receptor, for example a genomic sequence or a cDNA sequence, said sequence being introduced into the cells of the animal, at a early stage of embryonic development, for example not exceeding 8 cells, the crossing of animals resulting from the development of the embryos thus modified, under conditions allowing the production of a transgenic animal expressing jointly in its somatic cells and / or in its cells germ cells, human CD4 and CD26 receptors.

Other features and benefits of 1 ^• invention appear in the examples and the following figures.

These figures have the following meaning: Figure 1 - The tripeptide IPI does not affect the binding of gpl20 to CEM cells.

CEM cells (5.10 °) were incubated (37 ° C, 1 hour with gpl20 labeled with ¹²⁵ I (50 ng; 10 Ci / mg) in the absence (lines-) or in the presence of 20 mM IPI (line DipA) and OKT4, 0KT4A, 1F7, 110/4 and 71/10 monoclonal antibodies. The cells were then washed with PBS and the cytoplasmic extracts were analyzed by SDS PAGE. Recombinant gpl20 was radioiodinated with Bolton reagent Hunter as described by Krust B. et al in AIDS Res. Hmm. Retroviruses (1993 vol 9,

1081-1084). An autoradiogram is presented.

No apparent internalization of gpl20 took place under these experimental conditions. Treatment of the cells with trypsin led to the loss of more than 95% of gp120 labeled with ¹²⁵ I, bound to the cells. The binding of gpl20 to CEM cells was specific since the antibody 0KT4A directed against the binding site of gpl20 on the CD4 molecule completely abolished the binding.

Figure 2 - DPPIV-like peptidase activity on the surface of CD4 positive cells.

CEM cells MOLT4 and U937 (5.10 °) in a peptidase buffer (Hong W, et al. J. Cell Biol. 1990;

111: 323-328) containing either 0.5 mM GP-pNa (for DPPIV peptidase activity) or R-pNa (for activity

Arg-peptidase) were incubated at 37 ° C for 30 minutes, 1, 2, 3 and 4 hours.

The ordinate gives the values of the optical density measured at 405 nm.

Figure 3 - DPPIV-like peptidase activity on the surface of CD4 negative human and murine cells.

Human (HeLa and SK-N-MC) and murine (NIH / 3T3) cells were tested for DPPIV-like peptidase activity under the conditions of FIG. 2.

Figure 4 - Consensus sequences and sequences of different isolates from the V3 loop of HIV or SIV. Figure 5 - Co-culture of H9 / HIV-1 cells with murine T cells expressing human CD4 on the one hand, and human CD4 and CD26 on the other hand. Examples

1. GP dipeptidigues patterns in the loops of the HIV-1, HIV-2 and SIV isolates have the specificity required for DPPIV.

DPPIV manifests exopeptidase activity and endopeptidase activity characterized by the hydrolysis of a bond adjacent to a proline residue with a specific preference for GP-like dipeptides and also for AP, KP, RP, EP and DP (Nagatsu) dipeptides T, et al. Anal. Biochem. 1976; 74: 466-476) or tripeptides GPG, APG, KPG, RPG, EPG or DPG. The letter code was used to denote the amino acid residues of the peptides. According to this code, A represents alanine, P represents proline, K represents lysine, R represents arginine, E represents glutamic acid and D represents aspartic acid. The purified enzyme shows slightly different affinities, depending on the pH of the reaction (Nagatsu et al). For example, the relative activity of the enzyme at pH values equal to 7.0 and 8.7 with respect to XPp-nitroanilide peptides, from the highest to the lowest is as follows: pH 7.0: KP, GP, RP, AP, EP and DP pH 8.7: GP, AP, KP, RP, EP and DP Interestingly, the crown of the V3 loop of HIV- l contains the tripeptide GPG which can be cleaved by the action of DPPIV. In addition, at the N-terminal end of the V3 loop, there is a dipeptide motif RP which is conserved in all the HIV-1, HIV-2 and SIV isolates tested. In addition, the HIV-2 and SIV isolates have conserved RP or KP dipeptide motifs at the C-terminal end of the V3 loop (Table 1).

Conservation of RP, KP and GPG motifs

The dipeptide motifs RP and KP at the N- and / or C-terminal ends of the V3 loop are preserved in HIV-1, HIV-2 and SIV isolates. For example, in the case of HIV-1, the GPG motif is conserved in more than 90% of the HIV-1 isolates; the GPG motif remains unchanged in vivo for several years in a given individual despite other substitutions at the top of the V3 loop; the GPG motif is highly conserved among HIV-1 HIV-positive patients (Brown, AIDS 1991, 5: 535-542). In a study by Holmback et al, (J. Virol. 1993, 67: 1612-1619) on 30 hemophiliac patients with a CD4 cell number greater than or equal to 100 cells / mm ³ , 27 have a GPG motif and 3 have a conservative substitution of the APG type. It is noted that the AP-like motif comes just after the GP motif, as regards the specificity with respect to the DPPIV substrate; substitutions of the proline residue of the GPG motif to form GAG or GSG motifs generate virions whose infectious nature is suppressed. Furthermore, such mutations do not affect the expression of the envelope or its binding to the CD4 receptor, but abolish the induction of syncytia; substitutions of GPG for APG, GQG GVG and GPF generate envelope products whose capacity to induce syncytia is significantly reduced (Grimalia et al, J. Virol. 1992; 66: 1875-1883); in cell cultures in vitro, the GPG motif has been reported as a GQG mutant when HIV-1 infection is carried out in the presence of a specific monoclonal antibody directed against the V3 loop (Masuda et al J. Immunol. 1990; 145 : 3240-3246). The corresponding synthetic peptide GQG V3 also manifests a reduced affinity for the monoclonal antibody.

Conservation of the RP motif at the N-terminal end of the V3 loop among the HIV-1, HIV-2 and SIV isolates: the RP / KP motif in HIV-2 and SIV and the GP motif in the HIV-1 isolates are kept in more than 90% of isolates, which indicates that these dipeptide motifs in the V3 loop are under constant selection pressure and are essential for the infectious nature of the virus.

2. Experimental procedures for measuring the entry of HIV particles into cells.

The stock of HIV1-LAI isolates propagated on a CD4 positive T cell (CEM) cell line (Laurent AG, et al. J. Gen. Virol. 1990; 71: 2273-2281) was used in all the experiments. described. The virus was prepared in RPMI-1640 medium supplemented with 10% fetal calf serum and 2 μg / ml of Polybrene. The internalization of HIV particles was measured by a slightly modified technique described above (Harouse JM, et al. Science 1991; 253: 320-323). In this technique, the entry of HIV-1 particles into these cells was followed by the intracellular concentration of main core protein (p25) after elimination of the extracellular virus by treatment with trypsin. Briefly, CEM cells (5 × 10 ⁶ ) were suspended in 1 ml of HIV-1 preparation at a dose corresponding to 10 ⁶ TCID 50 per ml, leading to the production of more than 90% of cells producing HIV at days 3 and 4 after infection (pi or post-infection in English). The dose of virus suspension which, when used to inoculate each culture with a given number of tissue cultures, results in observable effects in 50% of these cultures.

After an hour of incubation at 37 ° C., the virus remaining adsorbed on the cells was eliminated by washing the cells first with PBS containing 2 mM EDTA and then by treatment with trypsin (2.5 mg / ml for 10 minutes at room temperature). Digestion was stopped by adding 10 times RPMI-1640 medium containing 10% (v / v) calf serum fetal. Cytoplasmic extracts were prepared by lysis of the cells in an extraction buffer containing 20 mM Tris-HCl, at pH 7.6, 0.15 M NaCl, 5 mM MgCl ₂ , 0.2 mM PMSF, 100 units / ml aprotinin and 0.5% Triton X-100. After centrifugation at 1000 g, the concentration of p25 in the supernatant was measured by an ELISA test. Under these experimental conditions, no significant entry of HIV occurred during the 1 hour incubation period at 4 ° C. This is normal since HIV can bind CD4 receptors at 4 ° C but it cannot enter cells. Furthermore, at 37 ° C, a considerable amount of HIV entered the cells. For example, in one of the experiments, the intracellular p25 concentrations at 4 ° C and 37 ° C were found at a height of 5 ". Attention: The printer does not have the font DA_MATH8a, replacement by Courier -3 and 185 - 24 pg for 10 ⁵ cells respectively It should be noted that only a very small proportion of the viral particles present are internalized in the cells.

This test for detecting the entry of HIV into CEM cells is highly reproducible (Krust B, et al. AIDS Res. Hum. Retroviruses 1993 in press). Consequently, it has been used here to investigate the various stages involved in the mechanism of the entry of HIV into cells.

3. Inhibition of the Entry of HIV-1 into CEM Cells by Peptides Corresponding to the V3 Loop

Peptides corresponding to the V3 loop have been described as capable of inhibiting more than 60% of the formation of syncytia during infection with HIV-1 (Koito A, et al. Intern. Immunol. 1989; 1: 613-618 ). To examine the effect of the V3 loop on the penetration of HIV particles into cells, different peptides corresponding to different regions of the HIV-1 surface protein (gpl20) and the HIV-1 transmembrane protein (gp41) were analyzed. The entry of HIV into permissive T cells of the CEM line was tested. The cells preincubated for 15 to 30 min. with the various peptides, were incubated with HIV-1LAI for one hour and the extracellular virus was eliminated by a treatment with trypsin according to the methods described in point 2. The results are summarized in Table 2 and show that the peptides of the V3 loop inhibits 88 to 96% of the penetration of HIV viral particles and this effect is specific since other peptides in gpl20 do not lead to a significant effect. In addition, the peptides corresponding to the top of the V3 loop also inhibit the entry of HIV particles. Interestingly, the peptide gp41 which contains GP- and RP-like motifs in its sequence (see Table 2a) leads to a 46% inhibition of the entry of HIV (Table 2).

In the process of entering HIV, it has been reported that the V3 loop is not required for binding of gpl20 to the CD4 receptor (Chiou SH, et al. AIDS Res. Hum. Retroviruses 1992; 8: 1611-1618 ) and that cleavage of the V3 loop may be necessary for the fusion process. The potential inhibition effect of V3 peptides on the entry of HIV into cells (Table 2), suggests that the V3 loop probably interacts with an additional receptor on the surface of the cells, which may also exhibit proteolytic activity. specific. The binding of gpl20 to the CD4 receptor can lead to conformational variations necessary to unmask or / and modify the conformation of the V3 loop, optimal conformation for the interaction with DPPIV / CD26. 4. Specific DPPIV inhibitors inhibit the penetration of HIV particles and abolish HIV infection.

The tripeptide IPI (also called diprotin A) isolated from the filtrates of a culture of Bacillus cereus is a specific inhibitor of DPPIV (Umezawa H et al J. Antibiot.; 1984; 37; 422-425). The effect of this inhibitor on the entry of HIV was tested at the same time as that of other peptides: VPL, GPA, GPGG, GGG, GP, KPR and RPGFSPFR (Table 3). The peptides were obtained from SIGMA and are purified by 99% HPLC. The tripeptide IPI has led to an inhibition greater than 90% of the entry of HIV-1 into the cells. The IPI tripeptide inhibition effect was dose dependent. Similarly, the KPR and RPGFSPFR peptides have led to an inhibition of the entry of HIV into CEM cells, greater than 90%. No apparent effect was observed with the peptides VPL, GPA, GGG and GP. Interestingly, the GPGG peptide having the GPG conserved motif at the top of the V3 loop exerted a significant inhibiting effect.

When infection with HIV-1 LAI of CEM cells was carried out in the presence of the peptide IPI (10 mM), no formation of syncytia was observed and the production of virus was inhibited by more than 95% (Table 4).

These results demonstrate that the tripeptide IPI is a specific inhibitor of HIV infection by preventing the entry of viral particles into cells.

The value of the inhibitory concentration at 50% of the entry of HIV into the cells was obtained by using 5.10 ⁶ cells at different concentrations of each peptide tested. 5. Inhibition of the penetration of HIV particles by specific DPPIV onoclonal antibodies

The entry of HIV-1 LAI particles was tested in the presence of various murine monoclonal antibodies: the monoclonal antibody 0KT4A (Ortho Diagnostics Systems) is directed against the CD4 receptor and recognizes the binding site of gpl20 (Sattentau QJ, et al. Cell 1988; 52: 631-633). the monoclonal antibody 0KT4 (Ortho Diagnostics Systems) recognizes an epitope close to the transmembrane domain of the CD4 receptor. This antibody has been described as not affecting the binding of gpl20 to the CD4 receptor (Sattenteau et al). the monoclonal antibody 110/4 (Genetic Systems, Seattle) is directed against the V3 loop and it completely neutralizes the infection by HIV-1. This monoclonal antibody has been described as not affecting the binding of gpl20 to the CD4 receptor (Kinney-Thomas E, et al. AIDS 1988; 2: 25-29).

- the BA5 antibody is directed against DPPIV was obtained from the company Immunotech in Marseille - France.

- the monoclonal antibody 1F7 specific for DPPIV has been described in the publication by Torimoto C (J. Immunol. 1989; 143: 3430-3439). the monoclonal antibody ALB1 is directed against the CALLA CD10 antigen (Immunotech Marseille) which is a neutral endopeptidase associated with the cell membrane (Barclay AN, et al. Facts Book. London: Harcourt Brace Jovanitch).

- the antibody CD11a (Immunotech) is directed against the alpha chain of the functional antigen-1 on lymphocytes (LFA-1) which is a member of the group of the family of adhesion molecules (Pardi R, et al. Immunol. Today 1992; 13: 224-230). - the monoclonal antibody CD18 (Immunotech) is directed against the β chain of LFA-1.

Table 5 gives the results of these experiments. The monoclonal antibodies 0KT4A and 110/4 led to an 85 to 93% inhibition of the entry of HIV-1 into the cells. The monoclonal antibody OKT4 led to an inhibition of 47% and this inhibition could be due to an indirect effect on the binding of gpl20 to the CD4 receptor (see Figure 1). The BA5 antibody inhibited the entry of HIV to a level greater than 60% while the 1F7 antibody led to an inhibition of 84%. The monoclonal antibodies ALB1, CD11a, and CD18 had no significant effect on the entry of HIV.

These results demonstrate that the specific antibodies directed against DPPIV have the capacity to block the entry of HIV and infection. This was then confirmed by the inhibition of 90% of the production of the virus when the infection was carried out in the presence of the antibody 1F7.

The monoclonal antibodies specific for the neutral endopeptidase CD10, the tyrosine phosphatase CD45 and the CD18 and CD11a antigens of the lymphocytes do not affect the entry of HIV.

6. The tripeptide IPI inhibits the entry of HIV at a stage following the binding of gp! 20 to the CD4 receptor

To study the mechanism by which the tripeptide IPI inhibits the entry of the virus, the binding to CEM cells of gpl20 labeled with ¹²⁵ I was studied, in the absence or in the presence of IPI and of the monoclonal antibodies OKT4, OKT4A, 1F7 and 110/4. The monoclonal antibody 71/10 directed against a protein kinase associated with the ribosomes (PKR) (Meurs EF, et al. Proc. Natl. Acad. Sci. USA 1993; 90: 232-236) was used as a control (FIG. 1 ). Under these experimental conditions, the addition of OKT4A antibodies made it possible to completely abolish the binding of the labeled gpl20 to CEM cells thus indicating that the binding was specific. The OKT4 antibody led to an inhibition of approximately 40% in agreement with the weak inhibition by this antibody presented in Table 5 of the entry of HIV. The 110/4 antibody against the V3 loop, which has been described as not affecting binding, has made it possible to completely abolish the binding between CD26 and gpl20. Interestingly, the tripeptide IPI which inhibits more than 90% of the entry of HIV into CEM cells (Table 3) did not at all affect the binding of gpl20 to these same cells (Figure 1). This last result indicates that the inhibitory effect of the IPI on the entry of HIV is an effect following the binding event between the gpl20 and the CD4 receptor, probably due to an interaction of the V3 loop with a component of the DPPIV / type. CD26 on the cell surface. The observation that the entry of HIV was also inhibited by the antibody IF7 (Table 5) agrees with the hypothesis put forward by the inventors that the component on the surface of the cell is indeed DPPIV.

The antibody IF7 made it possible to obtain an inhibition of more than 50% of the binding of gpl20 to CEM cells (FIG. 1), and in other experiments of more than 80% This effect probably results from induced indirect interference by this monoclonal antibody as is the case with the 110/4 antibody.

7. The tripeptide IPI inhibits DPPIV activity on the surface of CEM cells

The presence of IV-like DPP peptidase activity on intact CEM cells was examined using the chromogenic substrate Gly-Pro-p-nitroanilide (GP-pNA) as described in the publication Beauvois B, (Beauvois B, et al Eur. J. Immunol. 1992; 22: 923-930). The DPPIV activity observed by the production of pNA as a consequence of the cleavage of GP-pNA was significantly inhibited by the tripeptide IPI while the monoclonal antibody IF7 did not affect it (Table 6). As the tripeptide IPI and the antibody IF7 inhibit the entry of HIV into CEM cells (Table 3), these agents could therefore interfere with the entry of HIV according to various mechanisms: IPI would inhibit the peptidase activity of DPPIV whereas the 1F7 antibody would block a different site from DPPIV. Thus, two fields appear fundamental in DPPIV, concerning the implication of DPPIV in the mechanism of the entry of HIV into CEM cells. It should be noted that IPI could also act for the inhibition of binding of the glycoproteins of the HIV envelope to CD26.

The inhibitory dose of 50% of the concentration was also calculated to measure the inhibition of DPPIV activity. The results are reported in Table 6. The IC ₅₀ value for each peptide was calculated using 5.10 ° cells at different concentrations of each peptide, with the substrate GP-pNA. Interestingly, the peptides which inhibited the entry of HIV also inhibited the cleavage of the GP-pNA substrate.

These results demonstrated the specificity of these peptide inhibitors and confirmed the role of CD26 in its peptidase function, in the process of entry of HIV into cells.

The tripeptides IPI and KPK inhibited the entry of HIV into other cells such as the lymphoblastoid cells M0LT4, Jurkat and the monocytoid cells U397 as well as in CD4 * T lymphocytes freshly isolated and activated by PHA, at comparable concentrations. to those observed for CEM cells. 8. Inhibition of HIV-2 infection by the tripeptide IPI and the monoclonal antibody MAblF7 specific for DPPIV

The regions of the surface glycoprotein of HIV-2 and SIV which correspond to the V3 loop of the gp120 of HIV-1 are not well defined (Moore JP, et al. AIDS 1991; 17: 191-196). The peptides of the V3 loop of HIV-2 can be used to obtain weakly neutralizing antibodies (Bjόrling E, et al. Proc. Natl. Acad. Sci. USA 1991; 88: 6082-6086), while the V3 region of SIV does not seem to play a role in neutralization. Thus the V3 regions of HIV-2 and SIV are not potentially neutralizing epitopes like those of the HIV-1 isolates.

The V3 regions of HIV-2 and SIV do not contain the GPG consensus tripeptide at the top of the loop. On the other hand, all the HIV-2 and SIV isolates contain conserved dipeptides, RP near the N-terminal end and RP or KP near the C-terminal end of their respective V3 region (Clément GJ, et al. AIDS Res. Hum. Retroviruses 1991; 7: 3-16). Since such dipeptides can also be substrates for DPPIV-like peptidase activity (Nagatsu T, et al. Anal. Biochem. 1976; 74: 466-476), DPPIV could therefore be involved in the entry of HIV-2 and SIV in CD4 T cells. To verify whether DPPIV is involved in the entry of HIV-2, a highly divergent and cytopathogenic HIV-2 EHO isolate among the various HIV-2 isolates was used. CEM cells were infected in the absence or presence of specific DPPIV inhibitors, such as the tripeptide IPI and the monoclonal antibody 1F7 (Table 7). After 1 hour of infection, to allow entry of the HIV-2 particles, the cells were subjected to a treatment with trypsin to remove the extracellular virus and were cultured for test for virus production. The IPI peptide and the 1F7 antibody led to an inhibition greater than 75% (78% and 82% respectively) of the production of virus while the corresponding controls (VPL GGG and the antibody ALB1) do not produced no apparent effect (Table 7). Apart from inhibiting virus production, cultures of HIV-2 EHO did not develop a cytopathic effect when treated with IPI or the antibody 1F7.

9. HIV infection is specifically inhibited by the tripeptide IPI

Endopeptidase and exopeptidase inhibitors with various specificities have been used to investigate their effect on HIV-1 infection (Table 8). Known inhibitors of endopeptidases or exopeptidases such as aprotinin, leupeptin, antipain, pepstatin and bestatin had no effect on the entry of HIV into cells and on DPPIV activity -like. None of these inhibitors therefore produced significant inhibition of HIV infection. Under the same experimental conditions, the tripeptide IPI led to 90% inhibition.

10. DPPIV activity on the surface of different cell types

Para-nitroanilide (pNA) derivatives such as GP-pNA and RP-pNA were used to test for the potential presence of a DPP IV peptidase and an Arg-peptidase on the surface of receptor positive human cell lines CD4: CEM, M0LT4 and U937 (Figure 2). DPP IV-like peptidase activity was found on each of the 3 cell lines but at different levels, the M0LT4 cells giving rise to levels several times higher than those observed with CEM and U937 cells. This the difference is specific since the level of Arg-peptidase activity was comparable between these three cell lines. Therefore, MOLT4 cells may be more susceptible to HIV infection in accordance with various observations indicating an increase in the cytopathogenic effect of HIV in MOLT4 cells compared to the effect observed in CEM and U937 cells.

We also studied the variation of the DPPIV enzymatic activity according to whether it manifests in human cells or murine cells.

The 50% inhibitory concentration (IC ₅₀ ) of IPI was measured using extracts of human (HeLa) and murine cells (NIH 3T3) and GP-pNA as substrate.

DPPIV-like peptidase activity was measured either on the surface of intact cells (5 10 ⁶ ) or using cell extracts (25 μl) in a total reaction volume equal to 0.5 ml in a peptidase buffer containing 100 mM Hepes pH 7.6, 120 mM NaCl, 5 mM KC1, 1.2 mM MgS0 ₄ , 8 mM glucose, 1% BSA and either 0.5 mM GP-pNA or RP-pNA. The incubation was carried out at 37 ° C. for 2 to 4 hours and the reaction was stopped by the addition of 1M sodium acetate pH 4.5 (1 ml). After centrifugation at 12000 g for 5 min., The pNA production in the supernatant was measured by adsorption at 405 nm. For the preparation of cell extracts so as to follow the DPPIV activity, the cells lysed in buffer E (75 μl / 10 ⁷ cells) were kept at 4 ° C for 10 min before centrifugation at 12000 g for 10 min The supernatant diluted with one volume of buffer BI was then centrifuged once more at 12000 g for 10 min and the supernatant was stored at -80 ° C. Buffer E contains 20 mM Tris HC1, pH 7.6, 150 mM NaCl, 5 mM MgCl ₂ , 0.2 M PMSF, 100 units / ml aprotinin and 0.5% Triton X-100.

The BI buffer contains 20 mM Tris-HCl, 400 NaCl, 50 mM KC1, 1 mM EDTA, 0.2 mM PMSF, 100 units / ml 5 mM aprotinin _/ 3-mercaptoethanol, 1% Triton X-100 and 20% glycerol.

11. The mechanism of inhibition of the entry of HIV by the peptides IPI and KPR is a post-binding event.

To determine the mechanism by which peptide inhibitors inhibit entry of the virus into cells, the binding of ¹²⁵ I-labeled gpl20 to CEM cells in the presence of different monoclonal antibodies and different peptide inhibitors was studied (Figure 4) . The monoclonal antibody OKT4A completely abolished the binding of ¹²⁵ I labeled gpl20 to CEM cells, indicating that the binding was specific. On the contrary, the monoclonal antibody OKT4 only partially inhibited the binding, which was in agreement with the observation of the weak inhibition of the entry of HIV by this antibody. The monoclonal antibody MAb 110/4 specific for the V3 loop and which exhibited a very strong neutralizing titer (Goudsmit et al, Proc. Natl. Acad. Sci. USA 85, 4478-4482, 1988; De Jong et al, J. Virol. 66, 6777-6780, 1992; Chesebro et al., J. Virol. 6_S, 6547-6554, 1992; Linsley et al., J. Virol. 6 ^ 2, 3695-3702, 1988) abolished the binding ; this result illustrates the critical role that the V3 loop plays in the entry of HIV into cells. Partial inhibition of gpl20 binding has been observed with the monoclonal antibody 1F7, suggesting that the CD26 receptor is located in the vicinity of the CD4 molecule. The peptide inhibitors and KPR had no apparent effect on binding, indicating that their action inhibiting the entry of virus in cells is a post-binding event of the virus to the cell, likely due to the interaction of the V3 loop with a cell surface component exhibiting DPP IV-like enzyme activity. The observation that the entry of HIV was also inhibited by the monoclonal antibody against CD26, showed that this component on the surface of the cell, necessary for the entry of the virus, is the molecule CD26.

12. Infection of murine cells with HIV requires the expression of human CD4 and CD26 molecules.

Preliminary studies have indicated that the additional component on the surface of cells, the presence of which is necessary for the entry of HIV, is like the species-specific molecule CD4. Thus the expression of human CD4 cDNA transfected into human and murine cells makes human cells susceptible to HIV infection, unlike transfected murine cells. Although HIV particles bind to both human and murine cells expressing the human CD4 molecule, entry of the virus takes place only in human cells (Maddon et al, Cell, A1, 333-348 (1986) ; Ashorn, Berger, Moss, J. Virol 6_4, 2149-2156 (1990); Clapha, Blanc, Weiss, Virology 181, 703-715 (1991).

In a comparative study, the inventors showed that the petpidase DPPIV-like activity on the surface of human CD4 ^' and murine CD4 ^" cell lines was present. However, these petpidase activities on murine cells (such as NIH 3T3 and L929) were resistant to inhibition by the peptide IPI. In other studies using extracts of human (HeLa) and murine (NIH 3T3) cells, the inventors have demonstrated that the murine enzyme is significantly less sensitive to inhibition by the IPI peptide than the human enzyme (Table 10). For example, the inhibitory concentration (IC ₅₀ ) of IPI was at least 100 times higher for murine DPPIV-like activity compared to the human DPPIV-like activity tested on the GP-pNA or RP-pNA substrates. The activity of another peptidase such as arginine peptidase (Bauvois, Sancéau, Wietzerbin, Eur. J. Immunol. 22., 923-930, 1992) was at a similar level when the enzyme was of origin human or murine and was relatively insensitive to the activity of the IPI peptide. These results confirmed that the inhibitory effect of the IPI peptide is specific for the DPPIV-like peptidase activity of human origin. The different IC ₅₀ values obtained with the IPI peptide, necessary for the inhibition of DPP IV activity in human and murine cells, made it possible to formulate the hypothesis that the differences in the CD26 sequence between these species were responsible for the inability of HIV to infect murine cells expressing the human CD4 molecule. The inability of murine CD26 to serve as a co-receptor for the human CD4 receptor has been demonstrated by the experiments, the results of which are given in Table 11. The NIH 3T3 cells were transfected with plasmid vectors expressing the human CD4 receptor and the human CD26 receptor. either individually or together. The transfected cells were then contacted with HIV-1 LAI and virus production was measured by infection of the CEM cells. No significant virus production by cells expressing the human CD26 receptor alone or the human CD4 receptor alone was detected. In contrast, cells expressing both human CD4 and CD26 receptors produced infectious viruses. Addition of inhibitors of HIV entry into cells such as the tripeptide IPI and heparin (Krust et al, 1993, AIDS Res. Hum Retroviruses vol. 9, pp. 1081-1084) during contacting HIV with transfected cells expressing both CD4 and CD26 led to complete inhibition of HIV infection. The inhibitory action of IPI and heparin illustrates the specificity of viral infection in the transfected cells. Above all, these results confirm that CD26 is essential for the entry of HIV into cells expressing CD4.

By using other specific peptide inhibitors and a specific monoclonal antibody (Mab 1F7), the inventors have clearly demonstrated the role of CD26 in the mechanism of the entry of HIV into cells. The similar inhibitory effects obtained with different inhibitors against unrelated variants of HIV such as HIV-1 LAI and HIV-2 EHO indicated that the need for the presence of CD26 is a general phenomenon for different isolates of HIV-1 and HIV- 2. The observation that peptide inhibitors blocked the entry of HIV without affecting the binding of the SU glycoprotein to the CD4 receptor, suggested that the CD4 molecule only serves as an efficient attachment site of the virus on the cell surface which then allows the interaction between the V3 loop and the CD26 receptor. The binding of the virion's SU glycoprotein to the CD4 molecule can induce conformational modifications making the V3 loop more accessible. It was observed that the binding of the monoclonal antibody specific for the V3 loop to the surface glycoprotein of HIV-1 (gpl20) was increased by the presence of the soluble receptor CD4 (McKeating, Cordell, Dean, Balfe, Virology 191, 732 -742, 1992). It has been shown in the present invention that the entry of HIV-1 is significantly blocked by both the monoclonal antibody MAb 1F7 and the tripeptides IPI / KPR whereas the peptidase DPP IV activity was inhibited only by the tripeptides in question. The MAb 1F7 antibody had no apparent effect on DPP IV activity (Tanaka et al, Proc. Natl. Acad. Sci. USA 90, 4586-4590, 1993). Furthermore, the same tripeptides did not affect the binding, while the monoclonal antibody MAb 1F7 exerted a partial inhibition effect. These two types of inhibitors can therefore interfere with the process of entry of HIV-1 into cells, by two independent mechanisms: IPI / KPR inhibit the catalytic site involved in DPP IV activity while the antibody MAb 1F7 affects the site for recognition of gpl20 by the CD26 receptor.

Material and method corresponding to the tests reported in Table 11

NIH 3T3 cells were transfected with plasmid vectors expressing the CD4 and CD26 receptors, either independently or together, by the calcium phosphate co-precipitation technique. According to this technique, the NIH 3T3 cells (1.2 × 10 ⁶ ) were spread out in 25 cm ² flat flasks and transfected three days later. The culture medium (Dulbecco) was replaced with fresh medium and after one hour, 10 μg of each plasmid (pLXN expressing the human CD4 receptor; pKG5 expressing the CD26 receptor) alone or mixed with the other plasmid was precipitated with cells by the calcium phosphate co-precipitation technique. After 48 hours, the transfected cells were washed with the medium containing 2 mM EDTA followed by another washing with the culture medium. The cells were then brought into contact with HIVl LAI (corresponding to 0.5 μg of p25) for 6 hours in the absence or in the presence of inhibitors of the entry of HIV (IPI, heparin). The cells were first washed in a medium containing 2mM EDTA then trypsinized to eliminate the extracellular virus. The cells were then re-spread in 75 cm ² flat flasks with fresh culture medium and incubated at 37 ° C for 24 hours. 1 ml of aliquot of each supernatant was then used to infect CEM cells (5 10 ⁶ ). The production of HIV1 (p25, ELISA) in CEM cell cultures was measured 7 and 11 days later in the supernatant. The plasmid expressing CD4 has been described by Maddon PJ et al (Cell 47: 333-348). The plasmid pKG5-CD26 has been described by Fleischer B. et al, Cellul Immunol. 146, 249-260, (1993).

The results presented here show the necessity of the presence of CD26 for the entry of HIV into cells and the fact that cells producing HIV in in vitro cultures are doomed to die. The SU-TM complex of the HIV envelope plays a key role in the entry of viral particles into cells and when expressed at the level of the membrane of infected cells, it is responsible for initiating the phenomenon of apoptosis by interaction with at least the CD4 molecule.

The involvement of CD26 in viral entry and infection has been indirectly illustrated by several previous studies. In vitro in fresh CD4 ⁺ T lymphocytes, viral entry takes place only after cellular activation, a process associated with an increase in expression of the CD26 surface antigen. Furthermore, observations in individuals infected with HIV have shown in vivo a selective decrease in CD4 ⁺ T cells expressing CD26 (De Pasquale A. et al Acta Haemat. 81, 19-21 (1989); Valle-Blazquez M. et al, J. Immunol. 149, 3073-3077 (1992); Vanha G. et al, J. AIDS 6, 749-757 (1993)). Such observations corroborate our results thus demonstrating the need for CD26 for entry and the fact that infected cell cultures are doomed to die.

The HIV envelope gpl20 / gp41 complex plays a key role in particle entry, and when this complex is expressed on the surface of infected cells, it is responsible for the initiation of apoptosis through interaction with the CD4 molecule (Terai C. et al, New Concepts in AIDS pathogenesis L. Montagnier, ML. Gougeon Eds. 1993, pp. 41-58). By analogy with the mechanism of entry of HIV described here and the previous observations indicating that CD26 is involved in the transduction of cellular activation signals (Haffer DA et al 1989, J. Immunol. 142, 2590-2596) CD26 is involved in the mechanism of induction of apoptosis by the gpl20 / gp41 complex.

The development of AIDS in HIV positive individuals is correlated with the virus load in the blood and lymphoid tissues. The drugs currently available to treat AIDS do not make it possible to eradicate the HIV genome from infected cells. The demonstration that CD26 is required for viral entry provides the means to develop new inhibitors of viral infection. The inhibition of the entry of HIV-1 and HIV-2 by the tripeptides IPI and KPR gives the possibility of developing simple but specific inhibitors which could block the function of CD26 and therefore be effectively used as therapeutic agents for patients with AIDS.

Construction of lineages

In a line of mouse T lymphocytes expressing human CD4 (U. Blank et al., Eur. J. Immunol. 23, 3057, 1993), the inventors have demonstrated that after transfection of the cells with a plasmid expressing Human CD26, HIV-1 enters these cells. The addition of AZT during infection of human CD4 and CD26 expressing T cells is blocked by AZT. Although cells are infected, virus production is very low due to restrictions on the transactivation of HIV LTR. In addition, the functioning of TAT and REV transactivators in these non-human cells has been described to be blocked due to their interaction with certain proteins which may be species specific.

Using the murine T cell line expressing human CD4 and transfected with a plasmid expressing human CD26, clones of these mouse cells expressing human CD4 and CD26 were obtained. Because of the restriction of HIV replication in mouse cells, the clones were used in a test to assess their ability to fuse with H9 cells chronically infected with HIV-1 (H9 / HIV-1). These latter cells expressing the envelope glycoproteins of the virus could be used as effector cells to interact with CD4 ⁺ cells and initiate fusion of cell membranes, thus forming ultinucleated cells or syncytia. The formation of syncytia (ie cell / cell fusion) like the entry of HIV (ie, virus / cell fusion) requires the CD4 receptor and also another cell surface component which is suggested to be specific of the species.

The photo (FIG. 5) shows several images of co-culture of H9 / HIV-1 cells with murine T cells expressing on the one hand human CD4 and on the other hand CD4 and CD26 together after 24 h of commencement of co-culture . To facilitate fusion, cell aggregation was obtained by the addition of Wheat Germ Agglutinin (WGE: 10 μg / ml). In this FIG. 5, the mouse cells expressing human CD4 and CD26 fuse with H9-HIV cells to form syncytia and sloshing which are the two typical characteristics of the cytopathogenic effect observed in human cells infected with the HIV virus. The formation of syncytia and bloating is not observed in co-cultures with mouse cells expressing only human CD4. These observations can be used as further evidence for the role of the CD26 antigen in the fusion mechanism initiated by the interaction of HIV envelope glycoproteins with the CD4 receptor.

Claims

1. Use for inhibiting the entry of retroviral particles of a human retrovirus of HIV type, in particular HIV-1 or HIV-2, in target cells of a patient infected with this HIV retrovirus, of a compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected by this retrovirus and on the other hand, the glycoproteins of the envelope of this HIV retrovirus.

2. Use of a compound according to claim 1, for inhibiting the formation of syncytia in a patient infected with a human retrovirus of HIV type.

3. Use of a compound according to claim 1 or claim 2, for inhibiting the induction of cellular apoptosis in a patient infected with a human retrovirus of the HIV type.

4. Use according to any one of claims l to 3, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, modifies the interaction between the CD26 receptor and the peptide sequence called "V3 loop "of the outer envelope glycoprotein of an HIV retrovirus.

5. Use according to any one of claims 1 to 4, characterized in that the compound capable of modifying the interaction between the CD26 receptor and the glycoproteins of the envelope of the HIV retrovirus, has the property

- to inhibit the recognition and / or the binding between the envelope glycoproteins and the CD26 receptor, and / or - To inhibit the capacity of the CD26 receptor, to cleave a peptide bond after a motif of the XP type in which X is preferably a residue of an amino acid chosen from the amino acids Gly, Ala, Lys, Arg, Glu and Asp.

6. Use according to any one of claims 1 to 5, characterized in that the capacity of the compound to modify the interaction of the CD26 receptor with respect to the glycoproteins of the envelope of the HIV retrovirus, is determined by the test including the following steps:

- detection of HIV proteins.

7. Use according to claim 6, characterized in that the test comprises the elimination of the extracellular virus and the quantification of the intracellular virus by:

- treatment of cells to eliminate the remaining extracellular retroviruses, for example by controlled digestion with trypsin, - if necessary, culturing cells for two or three days, preparing the cytoplasmic extracts by treating the cells with an extraction buffer, for example with a buffer containing 20 mM Tris-HCl, at pH 7.6, 0.15 M NaCl, 5 mM MgCl ₂ , 0.2 mM PMSF, 100 units / ml aprotinin and 0.5% Triton X-100,

- And if necessary, the measurement of the core protein concentration (core) of the HIV or SIV retrovirus, obtained in the supernatant, for example by an ELISA test.

8. Use according to any one of claims 6 or 7, characterized in that the step of bringing into contact a human HIV retrovirus, in particular HIV-1 or HIV-2, or a simian retrovirus SIV, in an amount equal to TCID 50 is produced with cells comprising on their surface CD4 and CD26 receptors.

9. Use according to any one of claims 1 to 8 characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a retrovirus human type HIV, in particular type HIV-1 or HIV-2 and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, is an inhibitor of peptide or non-peptide nature.

10. Use according to claim 9, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the envelope glycoproteins of this HIV retrovirus is capable of modifying the interaction between the CD26 receptor and the surface envelope glycoprotein.

11. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is an antibody directed against the CD26 receptor.

12. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a monoclonal antibody directed against an epitope of the CD26 receptor comprising all or part of the GWSYG sequence.

13. Use according to any one of claims 1 to 12, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and, on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a monoclonal antibody directed against an epitope of the CD26 receptor such as the antibody complex CD26 formed, neutralizes the catalytic activity of the CD26 receptor, or an epitope which does not affect catalytic activity.

14. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is an antibody directed against an epitope of the V3 loop of an HIV or SIV retrovirus containing the Gly-Pro (GP) motif or a GP type motif such as an Ala-Pro (AP), Lys-Pro (KP), Arg-Pro (RP), Glu-Pro (EP) or Asp-Pro motif (DP).

15. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is the IPI tripeptide or the KPR tripeptide or a peptide having in its structure the conformation of the tripeptide IPI or of the tripeptide KPR and carrying the inhibitory function of the peptide IPI and in particular a peptide or a polypeptide having in its structure a peptide exhibiting symmetry at the level of its amino acid sequence, the center of symmetry of this sequence being preferably consisting of a native or modified Proline residue.

16. Use according to claim 15, characterized in that the compound capable of modifying the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of the HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a peptide or a polypeptide containing the sequence carrying the inhibitory function of the IPI peptide and possibly having symmetry at the level of its amino acid sequence, the center of symmetry of this sequence preferably being constituted by a Proline residue, said peptide or polypeptide being coupled to a molecule carrier having the capacity to protect the peptide against the action of proteases in vivo.

17. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus is a soluble form of the CD26 receptor, in particular in that it is of the CD26 receptor lacking its transmembrane sequence.

18. Use according to any one of claims 1 to 10, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, contains a recognition motif contained in a substrate of the enzyme DPPIV, or a motif having a conformation sufficiently close to that of the above recognition motif, to be recognized by the enzyme DPPIV and to form with it an enzyme-substrate type complex.

19. Use according to claim 15, characterized in that it comprises, as active principle, a compound containing a recognition motif chosen from GP, RP, KP, EP or DP motifs, or from GPG, RPG motifs, KPG, EPG or DPG, the aforementioned motifs being either in the structural context of the peptide environment of the V3 loop of the HIV or SIV envelope glycoproteins, or in another context.

20. Use according to claim 18 or 19, characterized in that the recognition pattern is modified so as to preserve it in vivo from peptidase activities and in particular to prevent the enzymatic cleavage activity normally exerted by DPPIV on this motif.

21. Use according to claim 20, characterized in that the modification of the recognition motif corresponds to a modification of the peptide bond preceding or following the Proline residue so that this bond is resistant to cleavage by DPPIV.

22. Use according to any one of claims 18 to 21, characterized in that the compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human retrovirus of HIV type, in particular of HIV-1 or HIV-2 type and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, is associated with a heterologous or polyerized structure so as to promote its activity inhibition or prevention of infection by an HIV or SIV retrovirus in vivo.

23. Use according to claim 22, characterized in that the active principle is included within a synthetic matrix of peptide type, in particular to form a structure of MAP type.

24. Use according to claim 22, characterized in that the compound constituting the active principle is in polymerized form.

25. Use according to claim 18, characterized in that the compound constituting the active principle is in the form of a recombinant molecule, for example in the form of a fusion protein with an antigen such as HBsAg.

26. Use according to any one of claims 18 to 25, characterized in that the active principle is a peptide containing a sequence chosen from GPGRAF, KRPGNK, RPGNK, KRPRQ or EPGKN.

27. Method for the in vitro screening of compounds capable of modifying the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV type retrovirus, in particular of the type HIV-1 or HIV-2 and on the other hand, the glycoproteins of the envelope of this HIV retrovirus, characterized in that it comprises the test comprising the following steps:

- detection of HIV proteins.

28. The method as claimed in claim 27, characterized in that the step of bringing a human HIV retrovirus, in particular HIV-1 or HIV-2, or a simian SIV retrovirus into contact, in an amount equal to the TCID 50 is carried out with cells comprising on their surface CD4 and CD26 receptors.

29. Method for the in vitro screening of compounds capable of modifying the catalytic activity of a CD26-type receptor present on the surface of cells of a patient infected with a human-type retrovirus HIV, in particular of HIV-1 or HIV-2 type with respect to the glycoproteins of the envelope of this HIV retrovirus, characterized in that it comprises the following stages:

- bringing cells carrying the CD26 receptor on their surface, for example CEM cells, with a human retrovirus HIV-1 or HIV-2, or a simian retrovirus SIV, with a determined quantity of a test compound, has previously verified the ability to cleave an XP motif in an X-Pro-p-nitroanilide sequence in which X is a residue of an amino acid preferably chosen from the amino acids Gly, Ala, Lys, Arg, Glu and Asp, or a compound with a similar function

- incubation of the cells at 37 ° C. for a sufficient time and under conditions allowing the catalytic reaction between the CD26 receptor and the surface envelope glycoprotein of the HIV or SIV retrovirus,

- centrifugation of cells,

- the determination of the presence and if necessary of the protein concentration of the HIV or SIV retrovirus, obtained in the supernatant, for example by an ELISA test.

30. Compound capable of inhibiting infection in vivo with a human HIV retrovirus, in particular a retrovirus of the HIV-1 or HIV-2 type, characterized in that it is a compound capable of modifying or inhibit the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with an HIV retrovirus and, on the other hand, the glycoproteins of the HIV envelope, said compound being

- or a monoclonal antibody directed against an epitope comprising all or part of the GWSYG sequence

- either an antibody directed against an epitope of the V3 loop and / or other domains of the envelope glycoproteins of an HIV or SIV retrovirus containing the Gly-Pro-Gly (GPG) or Ile-Pro-Ile (IPI) motif or a GP type motif such as an Ala-Pro (AP), Lys-Pro (KP), Arg-Pro (RP) motif, Glu-Pro (EP) or Asp-Pro (DP), either a peptide having in its structure the conformation of the tripeptide IPI and in particular a peptide exhibiting symmetry at the level of its amino acid sequence, the center of symmetry of this sequence preferably being constituted by a native or modified Proline residue,

- Or a compound containing a recognition motif contained in a substrate of the DPPIV enzyme, or a motif having a conformation sufficiently close to that of the above recognition motif, to be recognized by the DPPIV enzyme and to form with it a complex of enzyme-substrate type.

31. Compound according to claim 30, characterized in that it is coupled to a carrier molecule having the capacity to protect it against the action of proteases in vivo.

32. Use of the monoclonal antibody 1F7 to modify the interaction between, on the one hand, a CD26 type receptor present on the surface of cells of a patient infected with a human HIV retrovirus, in particular of the HIV-1 type or HIV-2, and on the other hand the envelope glycoproteins of this retrovirus.

33. Use of peptides of IPI type or of molecules having the same inhibitory function with respect to HIV or SIV, of peptide or non-peptide nature, to modify the interaction between, on the one hand, a receptor of the CD26 type present at the cell surface of a patient infected with a human HIV retrovirus, in particular of the HIV-1 or HIV-2 type, and on the other hand the envelope glycoproteins of this retrovirus.

34. Composition comprising an anti-CD4 antibody and a CD26 receptor inhibitor according to any one of claims 30 or 31.

35. Compound according to claim 30, characterized in that the recognition pattern meets the definition given in any one of claims 19 to 26.

36. Compound according to any one of claims 30 or 35, characterized in that it is either associated with a heterologous structure for example to form a structure of MAP type or a recombinant protein, or is polymerized.

37. Recombinant cell, characterized in that it is capable of jointly expressing the human CD4 and CD26 receptors.

38. Recombinant cell according to claim 37, characterized in that it is a eukaryotic cell, in particular a human cell, for example a HeLa cell or a murine cell, for example a NIH 3T3 cell.

39. Transgenic animal characterized in that its somatic cells and / or its germ cells comprise a sequence coding for the human CD4 receptor and a sequence coding for the human CD26 receptor, under conditions allowing the joint expression of these two receptors.

40. An animal according to claim 39 as obtained by introduction into the cells of the animal at a stage of early embryonic development, for example not exceeding 8 cells, of a sequence coding for the human CD4 receptor and of a sequence coding for the human CD26 receptor, under conditions allowing the joint expression of these two receptors.

41. Transgenic animal characterized in that it is a descendant of a transgenic animal according to claim 39 or claim 40.

42. Transgenic animal according to any one of claims 39 to 41, characterized in that it is a mammal, for example a rodent for example a mouse or a monkey.

43. Process for obtaining a transgenic animal comprising in its somatic cells and / or in its germ cells a sequence coding for the human CD4 receptor and a sequence coding for the human CD26 receptor, under conditions allowing joint expression of these two receptors comprising the steps of:

The introduction into another animal of the same species of a sequence coding for the human CD26 receptor, for example a genomic sequence or a cDNA sequence, said sequence being introduced into the cells of the animal, at a early stage of embryonic development, for example not exceeding 8 cells, the crossing of animals resulting from the development of the embryos thus modified, under conditions allowing the production of a transgenic animal expressing jointly in its somatic cells and / or in its cells germ cells, human CD4 and CD26 receptors.

44. Vaccine for protection against infection by an HIV or SIV retrovirus, characterized in that it comprises the active principle as defined in any one of claims 1 to 26 or the compound according to claim 30, under conditions allowing the production of neutralizing antibodies against HIV infection or SIV.