FR2707170A1 - Expression of CD4 and CD26 receptors in recombinant cells, CD26 receptor inhibitors. - Google Patents

Abstract

The subject of the invention is a pharmaceutical composition, characterized in that it comprises, as active principle, 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 aforesaid recognition motif, to be recognized by the DPPIV enzyme and to form with it an enzyme-substrate type complex. The invention also relates to recombinant cells capable of expressing human CD4 and CD26 receptors.

Description

Expression of CD4 and CD26 receptors in

  recombinant cells. CD26 receptor inhibitors.

  The present application relates to means for the identification and preparation of novel inhibitors of infection with a human HIV retrovirus. It concerns in particular cells and recombinant organisms capable of expressing both the

  CD4 human receptor and the human CD26 receptor.

  The human immunodeficiency virus (HIV) is the etiological agent of the 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 cells, monocytes, and macrophages by HIV requires the interaction of the env gene products of the virus with the cellular CD4 receptor (Klatzman D, et al., Nature

1984; 312: 767-768).

  The env gene of HIV encodes a precursor polyprotein which is cleaved by a cellular protease,

  leading to the production of extra glycoproteins

  Cellular or surface (SU) and transmembrane (TM) cells (Eiden LE, et al., Immunol.Today 1992; 13: 201-206). Surface protein, through non-covalent interactions with the transmembrane protein is exposed on the surface of cells

  infected 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 family of immunoglobulins and a carboxy-terminal region containing the transmembrane and

  cytoplasmic (Eiden et al). The first amino domain

  The terminal of the CD4 molecule appears to be the binding site of HIV surface proteins. Consequently, this binding induces a conformational change 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, prior to fusion, the surface protein is likely to undergo proteolytic cleavage modification, which in the case of HIV-1 surface protein (GP120) appears to take place at the third variable domain, commonly referred to as the "V3 loop", that is, the major neutralization domain (PND) (Moore JP, et al AIDS 1991;

: S21-S33).

  Various observations have so far suggested that the V3 loop plays a critical role in HIV-1 infection: 1) Naturally occurring neutralizing antibodies 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) V3 loop specific monoclonal antibodies do not affect binding of gp120 to the CD4 receptor but block the process fusion (Kinney-Thomas E, et al., AIDS 1988; 2: 25-29); 3) Mutations in the V3 loop generate virions whose infectious nature is reduced (Grimaila RJ, et al., J. Virol., 1992; 66: 1875-1883); 4) Mutations in the V3 loop modify the

  cellular tropism (Chesebro B, et al., J. Virol.

  1992; 66: 6547-6554) and elicit a change in the HIV phenotype as a syncytium-inducing or non-syncytium inducing virus (De Jong JJ,

  et al. J. Virol. 1992; 66: 6777-6780).

  Loop V3 contains about 36 amino acids whose cysteines at both ends are connected by a disulfide bridge forming a loop. The V3 loop contains conserved basic residues and furthermore the sequence in the vicinity of the bound cysteines, is similar in most isolates. (Clements GJ, et al AIDS Res, Hum Retroviruses 1991, 7: 3-16). Therefore, it seems that there is a selection pressure for

  keep the specific sequences in the V3 loop.

  Monoclonal antibodies against (15-mer) peptides representing the crown of the V3 loop have the ability to neutralize HIV-1 (Langedijk JPM, et al., J. Gen. Virol., 1991; 72: 25192526). ), this suggests that the amino acid residues of the top of the loop are involved in the interactions

functionalities 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 the purified gp120 could have been cleaved into fragments of 50 and 70 kDa by different proteases: trombin and tryptase cleave the tryptic site (GPGR - AFVT), whereas cathepsin E cleaves a chymotrypsin-like site

  (GPGRAF 4 VT) (Clements GJ, et al AIDS Res Hum.

Retroviruses 1991; 7: 3-16).

  The inventors have been 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 to the corresponding sequences. HIV-2 and SIV retroviruses. They observed that a very large number of isolates (more than 90%) identified by HIV-1 have a peptide motif consisting of

  amino acid sequence Gly-Pro-Gly (Glycine -

  Proline - Glycine or according to the one-letter GPG code) and more specifically a Gly-Pro pattern, preserved. 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 isolates identified from

  HIV-1, HIV-2 and SIV present at the N-terminus

  end of loop V3 a conserved dipeptide motif RP (Arginine Proline). Moreover, for HIV-2 and SIV, there is also a conserved dipeptide motif RP (Arginine-Proline), KP (Lysine-Proline),

  EP (Glutamic Acid Proline) or DP (Aspartic Acid)

  Proline), at the C-terminus of the V3 loop.

  Moreover, for HIV 2 and SIV, there is also a conserved dipeptide motif RP (Arginine - Proline) or KP (Lysine - Proline) at the C-terminus of the

V3 loop.

  Outside the V3 loop, in the case of HIV-1, there are several conserved motifs, for example the sequences IPI (Isoleucine-ProlineIsoleucine), GPC (Glycine-Proline-Cysteine), PPG (Proline-Proline-Glycine), located respectively at amino acids 185 and 208, SU and 411 of TM, relative to the consensus sequence of

Myers et al (1992).

  The inventors have investigated to what extent these preserved peptide motifs could be associated with infection with the HIV retrovirus. They demonstrated that these conserved motifs are susceptible to interaction with a molecule other than the CD4 receptor at the level of the cells.

  infectable by the HIV retrovirus, or SIV. -

  The inventors have thus identified a new mechanism involved in infection with the HIV or SIV retrovirus, which is involved in particular in the infection of lymphoid cells (whose cells

  T) patients infected with HIV retrovirus.

  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 the infection of

cells by the retrovirus.

  In the case of CD4- cell infection, for example SK-N-MC neuronal cells (Xi Ling Li et al. J. Virol.1990, 64: 1383-1387) or 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 the CD4- cells.

  The inventors have now demonstrated that a surface cellular protein interacts with the glycoproteins of the envelope of HIV or SIV, in particular at the level of the V3 loop and is capable of cleaving it. This cleavage takes place in one 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 exopeptidase activity and under certain conditions 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 pnitroanilide (para-nitroanilide), these peptides having an X-prop-nitroanilide sequence, to produce an X-Pro peptide and p-nitroaniline (Nagatsu T , et al Anal Biochem 1976; 74: 466-476) X being an amino acid residue Glycine (Gly), Alanine (Ala), Lysine (Lys), Arginine (Arg), Glutamic acid (Glu) and Acid

Aspartic (Asp).

  The CD26 antigen is widely expressed as a transmembrane glycoprotein of 110 to 120 kDa, which is identical to the cell surface serine protease called dipeptidyl peptidase IV. This enzyme, which is generally regarded 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 that this enzyme allows the cleavage of X-

  Β-polypeptides, where P is a Proline residue, when X is a free N-terminal amino acid (Kudo M, et al., J. Biochem., 1985; 97: 1211-1218). In addition, DPPIV has been described as capable of catalyzing a cleavage

  "endopeptidase-like", after a dipeptide pattern "GP-

  like "within synthetic peptides (Kenny AJ, et al., Biochem J. 1976; 155: 169-182) and to attach to collagen (Beauvois B, et al., I. Marcel Dekker (Eds.) New York, 1991) DPPIV binds collagen but no biological property outside of this ability to behave as a receptor for collagen has been

  evidence so far for this enzyme.

  In any case, the binding of DPPIV to collagen does not affect its enzymatic activity (Hanski C., et al., Exp.Cell Res., 1988; 178: 64-72), thus suggesting the existence of 'catching and catalytic in

different areas of DPPIV / CD26.

  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 a membrane anchoring domain, the 6 amino acids N. The end-cells forming a cytoplasmic domain and the carboxy-terminal end of the protein constituting the extracellular domain (Ogata S, et al., J. Biol Chem 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 lymphoid and non-lymphoid organs (Gorrel MD, et al Cell Immunol 1991, 134: 205-215). It is expressed on apical membranes of epithelial cells (brush borders and canalicular surfaces), endothelial cells, T cells, follicular cells, dendritic 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 various 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 low in resting T cells, but increases significantly after activation; for this reason, DPPIV / CD26 is considered as a T cell activation antigen. DPPIV expressions will be used interchangeably

or CD26 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 involvement in the entry of retrovirus into lymphocyte cells such as T cells, monocytes and macrophages, allows to define new ways to fight against infection with an HIV or SIV retrovirus, and in particular gives access to new inhibitors of

  infection of the cells 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 viral envelope SU and TM. This step concerning the interaction of the SU / TM complex with DPPIV / CD26 is essential for the

  HIV penetration 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 retrovirus of the HIV type, in particular of the HIV type. -1 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 fragment of this compound, capable of associating with

HIV envelope glycoproteins.

  The expression glycoproteins of the HIV envelope

  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 non-glycosylated forms of the glycoproteins of the envelope. Similarly, the expression "glycoproteins of the envelope" means that the interaction sought involves either one of these glycoproteins SU or TM, or 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 CD26 receptor for inhibiting infection with 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. Such compositions are particularly useful for treating an infection with an HIV or SIV retrovirus. They can also be implemented for the

vaccine preparation.

  Also within the scope of the invention are means for screening molecules capable of modifying the interaction between, on the one hand, a CD26-like 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.

  The invention therefore relates to a pharmaceutical composition characterized in that it comprises, as active principle, 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 retrovirus of the HIV type, in particular of the HIV-1 or HIV-2 type, and secondly,

  glycoproteins of the envelope of this HIV retrovirus.

  By pharmaceutical composition is meant the combination of one part of one or more compounds capable of having a treatment action against a pathogenic state or of preventing such a state, on the human body or animal, on the other hand constituents allowing the use, administration and / or preservation of this active ingredient in such a way that it

can exercise its action.

  A compound having, according to the definition given above, the ability to modify, that is, in particular to alter or prevent the structural or functional interaction between the CD26-type receptor present on the surface of cells of a patient infected with a human retrovirus of the 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 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 the recognition by the CD26 receptor of the glycoproteins of the envelope of an HIV retrovirus, or SIV, or of altering or to prevent the binding of this receptor with this glycoprotein or to alter or block the enzymatic function of the receptor with respect to peptide motifs of the X-Pro type, X being a chosen amino acid residue

  among the residues Gly, Ala, Lys, Arg, Glu, or Asp.

  Such an inhibitor can directly block the sites of the CD26 receptor 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 attaching to these sites. It can also act indirectly on these receiver functions by

  example by steric inhibition of the sites in question.

  The subject of the invention is therefore a pharmaceutical composition, characterized in that it comprises, as active principle, a compound capable of modifying the interaction between the CD26 receptor and the glycoproteins of the HIV retrovirus envelope: by inhibition of recognition and / or binding between the envelope glycoproteins and the CD26 receptor, and / or - by inhibiting the ability of the CD26 receptor, to cleave a peptide bond after an XP-type pattern in which X is a residue of a chosen amino acid

  among the amino acids Gly, Ala, Lys, Arg, Glu and Asp.

  In particular, these inhibitors include 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 the interaction with the molecule. DPPIV / CD26, for example by providing a substrate for enzymatic activity

DPPIV.

  Specific inhibition of DPPIV activity on the cell surface leads to decreased production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) by phorbol-stimulated mononuclear cells -ester and a reduced proliferative response to activated mitogen T-cell IL-2 (Schon 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 may be involved in the induction and activation of cytokines that control the

  lymphocyte proliferation (Beauvois et al; Schon).

  DPPIV is present on both quiescent CD4 + and CD8 + T cells and its expression is enhanced by T cell activation (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 +, memory cell marker and with the activation induced by the stimulation of CD2 or CD3. These functions are confirmed by the ability of anti-DPPIV antibodies, to modulate the surface expression of DPPIV. These antibodies also promote an increase in the proliferative response of T cells treated with anti-CD3 or anti-CD2 antibodies, which is preceded by an increase in the mobilization of Ca 2+ ions. DPPIV is physically associated with a membrane-bound, tyrosine-phosphatase-like protein CD45. (Torimoto Y., et al J. Immunol 1991: 147: 25142517) (Alexander D, et al., Immunol.

Today 1992; 13: 477-481).

  These observations suggest that DPPIV plays a role in the regulation of human T cell activation through its interaction with CD45, itself known to intervene in lymphocyte activation. The interaction of HIV or SIV retrovirus envelope glycoproteins with DPPIV / CD26 could alter the balance of transduced signals during lymphocyte activation and lead to apoptotic cell death (Callebaut C. and Jacotot E., 1992; Master's thesis, University Paris 11). Indeed, the interaction of envelope glycoproteins of HIV retroviruses with lymphocytes initiates a series

  of events leading to apoptosis.

  An advantageous pharmaceutical composition according to the invention is characterized in that the ability 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 comprising the following steps: contacting, on the one hand, cells comprising on their surface the CD26 receptor, on the other hand an amount equal to the TCID50, of an HIV human retrovirus, in particular HIV-1 or HIV-2, or a simian SIV retrovirus, incubating the aforementioned cells and the HIV or SIV retrovirus at 37 ° C. for a time sufficient to allow the retrovirus to penetrate into the cells, in the presence of a determined quantity of the test compound; cells for eliminating retroviruses absorbed on cells; extracellular virus elimination and quantification of intracellular virus; centrifugation for separating retroviral proteins, for example 100 0 g, and recovery of the supernatant,

  - the detection of HIV proteins.

  According to a particular embodiment of this pharmaceutical composition, the above-defined contacting step of an HIV human retrovirus, in particular HIV-1 or HIV-2 or a simian SIV retrovirus, in an equal amount. with TCID 50, is carried out with cells comprising on their surface, CD4 receptors and CD26 receptors. In a particularly interesting way, these receptors are species specific and preferably are the human CD4 and CD26 receptors. According to another particular embodiment of the invention, the ability of a compound used as an active principle in pharmaceutical compositions according to the invention to modify the interaction of the CD26 receptor with respect to the glycoproteins of the envelope of the HIV retrovirus, may be determined by the test comprising the following steps: 1-bringing into contact on the one hand cells containing on their surface the CD26 and / or CD4 receptors, on the other hand an amount equal to TCID50, an HIV human retrovirus, in particular HIV-1 or HIV-2, or a simian SIV retrovirus, 2-incubating the aforementioned cells and the HIV or SIV retrovirus at 37 C for a time sufficient to allow the retrovirus penetration into the cells, in the presence of a determined amount of the test compound, 3 the washing of the cells to eliminate the retroviruses absorbed on the cells, 4 the treatment of the cells to eliminate extracellular retroviruses. stants, for example by controlled digestion with trypsin, - the preparation of the cytoplasmic extracts by treatment of the cells with extraction buffer, for example with a buffer containing 20 mM Tris-HCl, 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 supernatant recovery, 7 the detection and, where appropriate, 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 trypsin-controlled digestion step and to test viral production after 2 or 3 days. Thus, after step 3, the cells can be incubated in the culture medium and the measured viral production, 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 a

HIV retrovirus.

  A preferred pharmaceutical composition according to the invention is 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 an HIV-type human retrovirus , 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 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 an HIV-type human retrovirus, 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 for example a

  antibody against the CD26 receptor.

  Such an antibody is, for example, capable of forming a binding with an epitope of the CD26 receptor so that the interaction with HIV or SIV envelope glycoproteins is inhibited either directly or indirectly, for example by

steric hindrance.

  According to another embodiment of the invention, the pharmaceutical composition is 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 the 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 monoclonal antibody directed against a CD26 receptor epitope comprising all or part of the sequence GWSYG (Glycine-Tryptophan-Serine-TyrosineGlycine) 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 may involve three amino acid sequences of the amino acid sequence of the CD26 molecule, one being the sequence GWSYG the others being respectively the HGT and 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 forming part of the active principle of a pharmaceutical composition according to the invention is advantageously such that the complex which it forms with the CD26 receptor alters or neutralizes the catalytic activity of the

CD26 receiver.

  By way of example of another type of active principle that is specific to the preparation of the pharmaceutical compositions of the invention, mention may be made of the antibodies directed against an epitope of the V3 loop of an HIV or SIV retrovirus containing the Gly-Pro motif. (GP)

  or a GP-like pattern such as an Ala-Pro (AP) pattern, Lys-

  Pro (KP), Arg-Pro (RP), Glu-Pro (EP) or Asp-Pro (DP).

  Another pharmaceutical composition according to the invention is 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 an HIV-type human retrovirus , 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 the IPI tripeptide or a peptide having in its structure the conformation of the IPI tripeptide and in particular having in its structure a peptide or a polypeptide containing a sequence bearing the inhibitory function of the IPI peptide and possibly having a symmetry at its amino acid sequence, the center of symmetry of this sequence being preferably constituted by a residue

Proline native or modified.

  According to another embodiment of the invention, an active ingredient of interest for the production of the pharmaceutical compositions is a compound containing a recognition unit contained in a substrate of the DPPIV enzyme, or a unit having a conformation sufficiently close to that of the aforesaid reason

  recognition, to be recognized by the enzyme DPPIV.

  Such a compound is capable of behaving as a substrate of the DPPIV enzyme and thus occupying the enzymatic site of the molecule, preventing the binding between the DPPIV molecule and the envelope glycoproteins.

HIV or SIV.

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

of the DPPIV.

  Certain compounds that meet this definition may also be suitable for inducing the production of neutralizing antibodies against infection.

by HIV or SIV.

  An interesting compound comprising a substrate recognition unit of the DPPIV molecule contains, for example, a peptide unit chosen from the sequences GP, RP, KP DP or EP, or a chosen motif.

  among the sequences GPG, RPG, KPG DPG or EPG.

  According to a first embodiment of the invention, this enzymatic substrate recognition motif is presented in a structural context analogous to the peptide environment existing at the level of the V3 loop of the external envelope glycoprotein of HIV or SIV. In this respect, it should be noted, for example, that the presence of the glycine residue after the proline residue contributes to the formation of a structure

  P-type elbow favorable for the exposure of proline.

  According to another variant, the aforesaid pattern is in a peptide environment different from that existing at

level of the V3 loop.

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

EPGKN.

  These peptides can be isolated from

  HIV or SIV antigens or chemically synthesized.

  The amino acid residues they contain can

  to be of laevorotatory or destrogyre configuration.

  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 may retain the capacity

  to be cleaved by the enzymatic action of the DPPIV.

  According to another embodiment of the invention, a pharmaceutical composition is characterized in that the recognition pattern is modified so as to prevent the enzymatic cleavage activity normally occurring.

exercised by the DPPIV on this ground.

  An appropriate modification can be made at the level of the next peptide bond and / or preceding the Proline residue of the recognition motif, in order to prevent the cleavage of this bond and therefore to

  decrease or eliminate the sensitivity to peptidases.

  The usual techniques for modifying

  peptide bonds can be implemented.

  In particular, the non-limiting

  changes in the peptide bond -CONH-

  leading to the formation of a reduced bond (CH2NH), retro inverso (NHCO), methylene-oxy (CH2-O), thiomethane (CH2-S), carba (CH2CH2), cetomethylene

  (CO-CH 2), hydroxyethylene (CHOH-CH 2) or Aza (N-N).

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

October 1990.

  These compounds described above for their capacity to constitute active principles, and in particular the compounds containing a unit of type GP, RP, KP, EP, DP or a tripeptide unit type GPG, RPG, KPG, EPG, DPG, have the quite remarkable property of being able to enter the composition of vaccines against infection by an HIV or SIV retrovirus although the sequences of the pattern of recognition are not

  necessarily accessible on the glycoprotein gp120.

  Their activity and in particular their ability to induce antibodies against the envelope glycoproteins may indeed occur regardless of the isolate of the HIV retrovirus (of the HIV1 or HIV2 type in particular) or SIV in question, since this activity is defined from conserved sequences between different isolates of the retrovirus HIV sequences being further conserved at

  course of time in a patient infected with the virus.

  If necessary, the active ingredient will be associated 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 complex term, promoting its properties, for example recognition of its target or its properties

antigenic or immunogenic.

  Thus, the active ingredient likely to form part of the composition of the pharmaceutical composition is advantageously accompanied or contained in a heterologous structure, or polymerized in such a way as to promote its activity of inhibiting or preventing infection by an HIV or SIV retrovirus. By way of example, this compound can be included within a synthetic peptide-type matrix, in particular to form a MAP-type structure. Such structures have been described as well as their method of preparation in Tam, JP (PNAS, USA, 85, 5409-5413, August 1988). The inclusion of the drug active ingredient within MAP structures has the advantage of promoting its inhibitory properties with respect to retrovirus infection.

HIV or SIV.

  According to another embodiment of the invention, the active principle 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 an HBsAg type antigen for example by insertion into the S or pre-S sequence of the antigen. The 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 an HIV-type human retrovirus, in particular type HIV-1 or HIV-2 and secondly, the glycoproteins of the envelope of this HIV retrovirus is a soluble form of the CD26 receptor, including the CD26 receptor lacking

of its transmembrane sequence.

  The subject of the invention is also 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 the 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: the bringing into contact of the one part of cells comprising on their surface the CD26 receptor, on the other hand an amount equal to the TCID50, of an HIV human retrovirus, in particular HIV-1 or HIV-2, or of a simian SIV retrovirus, incubating the aforementioned cells and the HIV or SIV retrovirus at 37 ° C. for a time sufficient to allow the retrovirus to penetrate 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, - the Elimination of extracellular virus and quantification of intracellular virus, - centrifugation for separating the retroviral proteins, for example at 1000 g, and recovery of the supernatant,

  - the detection of HIV proteins.

  According to a first embodiment of this method, screening is carried out after bringing 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. in particular, human receptors. Preferably, this process comprises the following steps: - bringing into contact, on the one hand, cells comprising on their surface the CD26 and CD4 receptors, on the other hand an amount equal to the TCID50, of a human HIV retrovirus , in particular HIV-1 or HIV-2, or a simian SIV retrovirus, - the incubation of the aforementioned cells and the HIV or SIV retrovirus at 37 C for a time sufficient to allow the retrovirus to penetrate into the cells, in the presence a specific amount of the tested compound, - the washing of the cells to eliminate the retroviruses absorbed on the cells, - the treatment of the cells to eliminate the remaining extracellular retroviruses, for example by controlled digestion with trypsin, - the preparation of the extracts cytoplasmic by treating the cells with extraction buffer, for example with a buffer containing 20 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 5 mM MgCl 2, 0.2 mM PMSF, 100 units / ml aprotinin and 0.5% Tr iton X-100, centrifugation for separating the retroviral proteins, for example at 1000 g, and recovery of the supernatant, detection and, if appropriate, measurement of the core protein concentration of the HIV or SIV retrovirus, obtained in the supernatant, for example by a

ELISA test.

  An alternative to this test is to culture the cells after the trypsin-controlled digestion step and to test the production.

viral after 2 or 3 days.

  Also within the scope of the invention, the compounds capable of inhibiting in vivo infection with 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 receptor of the CD26 type present on the cell surface 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 , except monoclonal antibody 1F7 peptides

IPI, KPR and RPGFSPFR not modified.

  Particularly advantageous compounds are

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 GWSYG, HGT, NNDI sequences of the CD26 molecule; an antibody directed against an epitope of the V3 loop of an HIV or SIV retrovirus containing the motif

  Gly-Pro (GP) or a GP-like pattern such as an Ala pattern

  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 may 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 IPI tripeptide and in particular a peptide or a polypeptide presenting a symmetry at its level. amino acid sequence, the center of symmetry of this sequence being preferably constituted by a native or modified Proline residue, - a peptide, a polypeptide with the exception of 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 GPG, KPG, RPG, EPG or DPG type, or a structural and / or functional analogue of this peptide or of this polypeptide such as a non-peptidic molecule reproducing the essential three-dimensional conformation of this peptide or this polypeptide as existing in the V3 loop of the outer envelope glycoproteins. HIV or SIV or if appropriate in another peptide environment; for example, the peptides GPGRAF, KRPGNK, RPGNK, KRPRQ or EPGKN; 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 external envelope glycoproteins of HIV or SIV; a nucleotide sequence coding for an enzymatic substrate recognition pattern 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 ability to protect them

  against the action of proteases in vivo.

  When it comes to peptides or peptide derivatives, they are advantageously modified to reduce or eliminate their sensitivity to proteases in vivo. Such changes have been

  described in the previous pages. The compounds

  above may also be associated with substances such as interleukin, 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. The subject of the invention is also the use, as active principle of a pharmaceutical composition, of a compound chosen from those which have been described above, capable of modifying the interaction between on the one hand, a receptor of the CD26 type present on 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 glycoproteins

envelope of this retrovirus.

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

  likely to be infected with HIV or SIV.

  The invention also relates to compositions containing several inhibitors of HIV or SIV retrovirus infection. 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 vis-à-vis HIV or SIV.

  By way of example are also within the scope of the invention, compositions containing several types of inhibitors of the interaction between CD26 and the outer envelope glycoproteins of HIV or SIV,

  selected from those described above.

  Such a composition may contain an anti-CD4 antibody and a CD26 receptor inhibitor, as described above. It may 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 an inhibitor

  CD26 receptor or a CD26 receptor inhibitor.

  According to a particular aspect of the embodiment of the invention, the compounds or active principles can be administered to the patient so as to be released with delay in the body; thus they can be associated with calcium phosphate or with aluminum hydroxide. Can also be used, to prepare inhibitors of the interaction between CD26 / DPP IV and HIV or SIV envelope glycoproteins, 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 Help Molecular Design 6, 1992, 159-174).

  The invention also relates to the use of the polypeptide sequences of Table 9, to prepare 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 glycoproteins

envelope of this retrovirus.

  The invention furthermore relates to a polyclonal or monoclonal antibody directed against an antigenic sequence of HIV or SIV, capable of modifying, in particular of inhibiting 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 type HIV-1 or HIV-2, and secondly the

  envelope glycoproteins of this retrovirus.

  The invention also relates to compositions

  pharmaceutical products or compounds among those described

  above, to prevent or treat cell apoptosis.

  Also within the scope of the invention are sequences of the V3 region of HIV envelope glycoproteins, characterized in that they have the capacity to induce eg 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 cells.

  murines such as NIH3T3 cells.

  These cells can be used for the in vitro screening of substances likely to behave 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 that have been described above.

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

excludes the man.

  Transformation of only the somatic cells of the animal makes it possible to obtain CD4 and CD26 receptor expression in this animal but excludes transmission to their offspring. On the contrary, the modification of the germinal cells allows the transmission of genes or coding sequences of interest,

  in the next 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 coding sequence for the human CD4 receptor and of a sequence coding for the human CD26 receptor, under conditions allowing expression

joint of these two receivers.

  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 early stages of embryonic development in order to ensure that in the developed animal the transformation of

  all or almost all of these cells.

  Various techniques are available for effecting the insertion of a gene or sequence into the cells of an animal embryo. In particular, mention is made of the publication of Brinster RL et al, PNAS,

  USA, vol. 82, pp. 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 that has been transformed to introduce the genes or sequences of interest. It could be

  also from 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

  may also use monkeys.

  The invention also relates to a method for obtaining a non-human transgenic animal comprising in its somatic cells and / or in its germinal cells a coding sequence for the human CD4 receptor and a coding sequence for the human CD26 receptor, under conditions allowing the joint expression of these two receptors comprising the steps of: the introduction of a coding sequence for 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 encoding the CD26 common receptor, for example a genomic sequence or a sequence cDNA, said sequence being introduced into the cells of the animal, at an early stage of embryonic development, for example not exceeding 8 cells, - the crossing t animals resulting from the development of the embryos thus modified, under conditions making it possible to obtain a transgenic animal expressing together in its somatic cells and / or in its germinal cells, the

CD4 and CD26 human receptors.

Other features and advantages of the invention appear in the examples and

following figures.

  These figures have the following meaning: Figure 1 - The IPI tripeptide does not affect the link

from gpl20 to CEM cells.

  CEM cells (5.106) were incubated (37 C, 1 hour with 125I-labeled gp120 (50 ng, 10 Ci / mg) in the absence (lines-) or in the presence of 20 mM IPI (DipA line) and The OKT4, OKT4A, 1F7, 110/4 and 71/10 monoclonal antibodies were then washed with PBS and the cytoplasmic extracts were analyzed by SDS PAGE.The recombinant gp120 was radioiodinated with Bolton Hunter reagent according to as described by Krust B. et al in AIDS Res, Hum Retroviruses (1993 Vol 9,

  1081-1084). An autoradiogram is presented.

  No apparent internalisation of gp120 occurred under these experimental conditions. Treatment of the cells with trypsin resulted in the loss of more than 95% of 1125 labeled gp120, bound to the cells. The binding of gp120 to CEM cells was specific since the OKT4A antibody raised against the gp120 binding site on the CD4 molecule

completely abolished the bond.

  Figure 2 - DPPIV-like peptidase activity & surface

CD4 positive cells.

  CEM cells MOLT4 and U937 (5.106) 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 Argpeptidase activity) 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 at the surface

  negative human and murine CD4 cells.

  The human (HeLa and SK-N-MC) and murine (NIH / 3T3) cells were tested for DPPIV-like peptidase activity under the conditions of Figure 2. Figure 4 - Consensus sequences and sequences of

  different isolates from the V3 loop of HIV or SIV.

Examples

  1. Dipeptide GP motifs in loops of HIV-1, HIV-2 and SIV isolates show specificity

required for the DPPIV.

  DPPIV exhibits 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 dipeptides (Nagatsu T, et al Anal Biochem 1976, 74: 466-476) or tripeptides GPG, APG, KPG, RPG, EPG or DPG. The one-letter code was used to designate 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 has slightly different affinities depending on the pH of the reaction (Nagatsu et al). For example, the relative activity of

  the enzyme at pH values of 7.0 and 8.7 vis-

  for the peptides of XPp-nitroanilide type, from the highest to the lowest is the following: 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-1 contains the GPG tripeptide which can be cleaved by the action of DPPIV. In addition, at the N-terminus of the V3 loop, there is a RP dipeptide motif which is conserved in all tested HIV-1, HIV-2 and SIV isolates. In addition, the HIV-2 and SIV isolates have retained RP or KP dipeptide motifs at the C-terminus of the V3 loop.

(Table 1).

  Conservation of RP, KP and GPG motifs

  The RP and KP dipeptide motifs at the N-ends

  and / or C-terminus of the V3 loop are conserved in the HIV-1, HIV-2 and SIV isolates. By way of example, in the case of HIV-1, the GPG motif is conserved in more than 90% of 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 seropositive patients (Brown, AIDS 1991, 5: 535-542). In a study by Holmback et al., (J. Virol., 1993, 67: 1612-1619), 30 haemophiliac patients with a CD4 cell count greater than or equal to 100 cells / mm3, 27 had a GPG pattern and 3 had a conservative substitution of type APG. 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. Moreover, such mutations do not affect the expression of the envelope or its binding to the CD4 receptor, but abolish the induction of syncytia; GPG substitutions in APG, GQG GVG and GPF generate envelope products whose ability to induce syncytia is

  significantly reduced (Grimalia et al., J. Virol.

  1992; 66: 1875-1883); in in vitro cell cultures, the GPG motif has been reported as a mutant in GHQ when the 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; : 3240-3246). The corresponding synthetic peptide GQG V3 also has a reduced affinity for

the monoclonal antibody.

  Retention of the RP motif at the N-terminus 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 preserved in more than 90% of the isolates, indicating that these dipeptide motifs in the V3 loop are under constant selection pressure and are essential for their character.

infectious virus.

  2. Experimental procedures for the measurement of the input

  HIV particles in cells.

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

in 50% of these cultures.

  After one hour of incubation at 37 ° C., the remaining virus adsorbed on the cells was removed by washing the cells first with PBS containing 2 mM EDTA and then trypsin treatment (2.5 mg / ml for 10 minutes). minutes at room temperature). Digestion was stopped by the addition of 10 times RPMI-1640 medium containing 10% (v / v) fetal calf serum. Cytoplasmic extracts were prepared by lysing the cells in extraction buffer containing 20 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 5 mM MgCl2, 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 ELISA. Under these experimental conditions, no significant HIV entry occurred during the incubation period of 1 hour at 4 C. This is normal since HIV can bind CD4 receptors to

  4 C but he can not enter the cells.

  In addition, 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 370 C were found at a height of 5 -LAttention: The printer does not have the DAMATH8a font, Courier -3 replacement and 185 - 24 pg for 105 cells respectively. It should be noted that only a very small proportion of the particles

  Viral diseases are internalized in the cells.

  This test for detecting HIV entry into EMC cells is highly reproducible (Krust B, et al., AIDS Res, Hum Retroviruses 1993 in press). As a result, it has been used here to investigate the different steps involved in the mechanism of

the entry of HIV into the 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 being able to inhibit more than 60% of the formation of syncytia during HIV-1 infection

  (Koito A, et al., Immunol 1989, 1: 613-618).

  To examine the effect of the V3- loop on the penetration of HIV particles in cells, different peptides corresponding to different regions of the surface protein (gp120) of HIV-1 and the transmembrane protein (gp41) of HIV-1 have been 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 different peptides, were incubated with HIV-1LAI for one hour and the extracellular virus was removed by trypsin treatment according to the modalities described in point 2. The results are summarized in Table 2 and show that the peptides of The V3 loop inhibits 88-96% of HIV virus particle penetration and this effect is specific since other peptides in gp120 do not lead to a significant effect. In addition, the peptides corresponding to the top of the V3 loop also inhibit the entry of the HIV particles. Interestingly, the gp41 peptide which contains GP- and RP-like motifs in its sequence (see Table 2a) leads to a 46% inhibition

HIV entry (Table 2).

  In the HIV entry process, it has been reported that the V3 loop is not required for gp120 binding to the CD4 receptor (Chiou SH, et al., Res Res Hum. Retroviruses 1992; 8: 1611-1618 ) and cleavage of the V3 loop might be necessary for the fusion process. The potential inhibitory effect of V3 peptides on HIV entry into cells (Table 2) suggests that the V3 loop probably interacts with an additional cell surface receptor, which may also exhibit proteolytic activity specific. The binding of gp120 to the CD4 receptor may result in conformational variations necessary to unmask or / and modify the conformation of the V3 loop, conformation- optimal

for interaction with DPPIV / CD26.

  4. Specific inhibitors of DPPIV 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). Peptides were obtained from SIGMA and purified by 99% HPLC. The tripeptide IPI led to obtaining a greater than 90% inhibition of the entry of HIV-1 into the cells. The inhibition effect of IPI tripeptide was dose dependent. Similarly, the KPR and RPGFSPFR peptides have led to an inhibition of the entry of HIV into the CEM cells, greater than 90%. No apparent effect was observed with the VPL, GPA, GGG and GP peptides. Interestingly, the GPGG peptide having the conserved GPG motif in the apex of the V3 loop exerted a significant inhibitory effect. When the HIV-1 LAI infection of the CEM cells was carried out in the presence of the IPI peptide (10 mM), no syncytia formation was observed and the virus production was inhibited by more than 95%.

(Table 4).

  These results demonstrate that the IPI tripeptide is a specific inhibitor of HIV infection by preventing entry of virus particles into cells. The value of the 50% inhibitory concentration of HIV entry into the cells was obtained using 5 × 10 6 cells at different concentrations.

of each peptide tested.

  5. Inhibition of the penetration of HIV particles by monoclonal antibodies specific for DPPIV The entry of the particles of HIV-1 LAI was tested in the presence of different murine monoclonal antibodies: the monoclonal antibody OKT4A (Ortho Diagnostics Systems) is directed against the CD4 receptor and recognizes the binding site of the gp120 (Sattentau QJ,

et al. Cell 1988; 52: 631-633).

  the OKT4 monoclonal antibody (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 gp120 to

CD4 receptor (Sattenteau et al).

  the 110/4 monoclonal antibody (Genetic Systems, Seattle) is directed against the V3 loop and completely neutralizes the HIV-1 infection. This monoclonal antibody has been described as not affecting the binding of gp120 to the CD4 receptor (Kinney-Thomas

E, et al. AIDS 1988; 2: 25-29).

  - the BA5 antibody is directed against the DPPIV has been

  obtained from the Immunotech Company 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 membrane-associated neutral endopeptidase

  cell (Barclay AN, et al., Facts Book.

London: Harcourt Brace Jovanitch).

  the CD11a antibody (Immunotech) is directed against the lymphocyte-functional antigen-1 alpha chain (LFA-1) which is a member of the adhesion molecule family (Pardi R, et al.

Immunol. Today 1992; 13: 224-230).

  the monoclonal antibody CD18 (Immunotech) is directed

against channel 3 of LFA-1.

  Table 5 gives the result of these experiments.

  Monoclonal antibodies OKT4A and 110/4 led to 85-93% inhibition of HIV-1 entry into the cells. The monoclonal antibody OKT4 led to a 47% inhibition and this inhibition could be due to an indirect effect on the binding of gp120 to the CD4 receptor (see FIG. 1). The BA5 antibody inhibited the entry of HIV at a level greater than 60% while the 1F7 antibody led to an inhibition of 84%. The monoclonal antibodies ALB1, CDlla, and CD18 did not

  no significant effect on HIV entry.

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

of the 1F7 antibody.

  Monoclonal antibodies specific for CD10 neutral endopeptidase, CD45 tyrosine phosphatase and CD18 and CD11a antigens for lymphocytes

do not affect the HIV entry.

  6. The IPI tripeptide inhibits HIV entry at a step following the binding of gp120 to the CD4 receptor. To study the mechanism by which the IPI tripeptide inhibits virus entry, the binding to GPL20 CEM cells was studied. labeled with 125I, in the absence or presence of IPI and OKT4, OKT4A, 1F7 and 110/4 monoclonal antibodies. Monoclonal antibody 71/10 directed against a ribosome-associated protein kinase (PKR) (Meurs EF, et al Proc Natl Acad Sci USA 1993, 90: 232-236) was used as a control (FIG. ). Under these experimental conditions, the addition of OKT4A antibodies completely abolished the binding of the labeled gp120 to the EMC cells indicating that the binding was specific. OKT4 antibody resulted in about 40% inhibition consistent with the low inhibition by this antibody shown in Table 5 of the HIV entry. The 110/4 V3 loop antibody which was described as not affecting the binding, completely abolished the binding between CD26 and gp120. Interestingly, the IPI tripeptide which inhibits more than 90% of HIV entry into CEM cells (Table 3) did not affect binding of gp120 at all 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 gp120 and the CD4 receptor, probably due to an interaction of the V3 loop with a DPPIV / type component. CD26 on the surface of the cell. The observation that the entry of HIV was also inhibited by the IF7 antibody (Table 5) is consistent with the hypothesis emitted by the inventors that the component on the surface of the cell is the same.

DPPIV.

  IF7 antibody resulted in more than 50% inhibition of gp120 binding to CEM cells (Figure 1), and in other experiments more than 1%. This effect probably results from indirect interference induced by this monoclonal antibody as

  this is the case with the 110/4 antibody.

  7. The IPI tripeptide inhibits DPPIV activity on the surface of the CEM cells The presence of a DPP IV-like peptidase activity on intact CEM cells was examined using the Gly-Prop-nitroanilide chromogenic substrate (GP-pNA) as described in Beauvois B, (Beauvois B, et al., J. Immunol 1992: 22: 923-930). DPPIV activity found by pNA production as a consequence of GP-pNA cleavage was significantly inhibited by the IPI tripeptide whereas IF7 monoclonal antibody did not affect it (Table 6). Since the IPI tripeptide and the IF7 antibody inhibit the entry of HIV into CEM cells (Table 3), these agents could therefore interfere with the entry of HIV according to different mechanisms: IPI inhibits the DPPIV peptidase activity while the 1F7 antibody would block a site different from DPPIV. Thus, two domains appear fundamental in the DPPIV, with regard to the involvement of the DPPIV in the mechanism of the entry of HIV in CEM cells. It should be noted that IPI could also act for inhibition of fixation of

  glycoproteins from the envelope of HIV to CD26.

  The 50% inhibitory dose of the concentration was also calculated to measure the inhibition of DPPIV activity. The results are reported in Table 6. The IC50 value for each peptide was calculated using 5.106 cells at different

  concentrations of each peptide, with the GP-substrate

  pNA. Interestingly, peptides that inhibited HIV entry also inhibited 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 method of entry of

HIV in the cells.

  The tripeptides IPI and KPK inhibited the entry of HIV into other cells such as MOLT4, Jurkat and U397 monocytoid cells as well as into freshly isolated and activated PHA-activated CD4 + T cells at concentrations comparable to those observed for

EMC cells.

  8. Inhibition of HIV-2 infection by the IPI tripeptide and the MAbF7 monoclonal antibody specific for DPPIV The regions of the HIV-2 and SIV surface glycoprotein which correspond to the V3 loop of the HIV-2 gp120 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 (Bjorling 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 epitopes

  neutralizers such as HIV-1 isolates.

  The V3 regions of HIV-2 and SIV do not contain the GPG consensus tripeptide at the top of the loop. In contrast, all HIV-2 and SIV isolates contain

  preserved dipeptides, RP near the N-terminus

  terminally and RP or KP near the C-terminus of their respective V3 region (Clements GJ, et al., Res Res Hum, Retroviruses 1991; 7: 316). Since such dipeptides may 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 test whether DPPIV is involved in the entry of HIV-2, a highly divergent and cytopathic HIV-2 EHO isolate among the different HIV2 isolates was used. CEM cells were infected in the absence or presence of specific DPPIV inhibitors, such as IPI tripeptide and monoclonal antibody 1F7 (Table 7). After 1 hour of infection, to allow entry of the HIV-2 particles, the cells were subjected to trypsin treatment to remove the extracellular virus and were cultured to test for virus production. The IPI peptide and the 1F7 antibody led to an inhibition of more than 75% (78% and 82%, respectively) of virus production, whereas the corresponding controls (VPL GGG and antibody ALB1) do not produced no apparent effect (Table 7). Apart from inhibition of virus production, HIV-2 EHO cultures did not develop a cytopathic effect when treated with IPI or

the 1F7 antibody.

  9. HIV infection is specifically inhibited by IPI tripeptide. Endopeptidase and exopeptidase inhibitors with various specificities were 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 have no effect on the entry of

  HIV in cells and on DPPIV-like activity.

  None of these inhibitors therefore produced significant inhibition of HIV infection. Under the same experimental conditions, the tripeptide IPI led

at 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 a peptidase on the surface of human cell lines positive for the CD4 receptor: CEM, MOLT4 and U937 (Figure 2). The DPP IV-like peptidase activity was found on each of the 3 cell lines but at different levels, the MOLT4 cells giving rise to levels several times higher than those observed with the CEM and U937 cells. This 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 agreement with different observations indicating an increase in the cytopathic effect of HIV in MOLT4 cells compared to the observed effect.

in CEM and U937 cells.

  The variation in DPPIV enzyme activity has also been studied as it occurs at the level of

  of human cells or murine cells.

  The 50% inhibitory concentration (IC50) of IPI was measured using extracts from human (HeLa) and murine (NIH 3T3) cells and GP-pNA as substrate. DPPIV-like peptidase activity was measured either on the surface of intact cells (5 106) or using cell extracts (25 μl) in a total reaction volume of 0.5 ml in a peptidase buffer containing 100 mM Hepes pH 7.6, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 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 production of pNA in the supernatant was measured by adsorption at 405 nm. For the preparation of the cell extracts so as to follow the DPPIV activity, the cells lysed in an E buffer (75 μl / 107 cells) were stored 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 an additional time at 12000 g for 10 min. and the supernatant was stored at -80 C. Buffer E contains 20 mM Tris HCl, pH 7.6, 150 mM NaCl, mM MgCl 2, 0.2 mM PMSF, 100 units / ml aprotinin and

0.5% Triton X-100.

  BI buffer contains 20 mM Tris-HCl, 400 NaCl, 50 mM KCl, 1 mM EDTA, 0.2 mM PMSF, 100 units / ml aprotinin 5 mM P-mercaptoethanol, 1% Triton X-100 and

% glycerol.

  11. The mechanism of inhibition of the entry of HIV by the peptides IPI and KPR is an event subsequent to

the link.

  To determine the mechanism by which peptide inhibitors inhibit virus entry into cells, the binding of 125I-labeled gp120 to CEM cells in the presence of different monoclonal antibodies and different peptide inhibitors was studied (Figure 4). The OKT4A monoclonal antibody completely abolished the binding of I-tagged gpl20 to the EMC cells, indicating that the binding was specific. In contrast, the OKT4 monoclonal antibody only partially inhibited binding, which was consistent with the observation of the low inhibition of HIV entry by this antibody. The mAb 110/4 monoclonal antibody specific for the V3 loop and which had 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 66, 6547-6554, 1992, Linsley et al., J. Virol 62, 3695-3702, 1988) abolished the linkage; this result illustrates the critical role that loop V3 plays in the entry of HIV into cells. Partial inhibition of gp120 binding was observed with monoclonal antibody 1F7, suggesting that the CD26 receptor is localized in the vicinity of the CD4 molecule. Peptide inhibitors and KPRs had no apparent effect on binding, indicating that their inhibitory action of virus entry into cells is an event subsequent to virus binding to the cell, probably due to the interaction of the V3 loop with a cell surface component exhibiting DPP IV-like enzymatic 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

CD26 molecule.

  12. Infection of murine cells with HIV requires

  the expression of human CD4 and CD26 molecules.

  Preliminary studies have indicated that the extra cell-surface component, which is needed for HIV entry, is like the species-specific CD4 molecule. Thus expression of the human CD4 cDNA transfected into human and murine cells makes the human cells susceptible to HIV infection, in contrast to the transfected murine cells. Although HIV particles bind to both human and murine cells expressing the human CD4 molecule, entry of the virus occurs only in human cells (Maddon et al, Cell, 47, 333-348 (1986) Ashorn, Berger, Moss, J. Virol 64, 2149-2156 (1990), Clapham, White, Weiss, Virology 181, 703-715;

(1991).

  In a comparative study, the inventors have shown that petpidase activity DPPIV-like at the surface

  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 IPI peptide.

  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 (IC50) of IPI was at least one-fold higher for murine DPPIV-like activity compared to human DPPIV-like activity tested on GP-pNA or RP-pNA substrates. The activity of another peptidase such as arginine peptidase (Bauvois, Sanceau, Wietzerbin, Eur J Immunol 22, 923-930, 1992) was at a similar level when the enzyme was of human origin. 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 IC50 values obtained with the IPI peptide, necessary for the inhibition of the DPP IV activity of 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 HIV's inability to infect

  murine cells expressing the human CD4 molecule.

  The inability of murine CD26 to co-receptor the human CD4 receptor was 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 the production of

  virus 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 have produced infectious viruses. The addition of HIV entry inhibitors to cells such as IPI tripeptide and heparin (Krust et al., 1993, AIDS Res, Hum Retroviruses, Vol 9, pp. 1081-1084) during implementation HIV contact with the 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.

  First and foremost, these results confirm that CD26 is essential for HIV entry into cells

expressing CD4.

  By using other specific peptide inhibitors and a specific monoclonal antibody (Mab lF7), the inventors have clearly demonstrated the role of CD26 in the mechanism of HIV entry into cells. Similar inhibitory effects achieved with different inhibitors against unrelated HIV variants such as HIV-1 LAI and HIV-2 EHO indicated that the need for the presence of CD26 is a general phenomenon for different HIV-1 and HIV-1 isolates. 2. The observation that peptide inhibitors blocked HIV entry without affecting the binding of SU glycoprotein to the CD4 receptor, suggested that the CD4 molecule serves only as an efficient site of attachment of the virus to the cell surface which then allows the interaction between the V3 loop and the CD26 receiver. The binding of the virion SU glycoprotein to the CD4 molecule can induce conformational changes making the V3 loop more accessible. It has been observed that the binding of the V3 loop-specific monoclonal antibody to the HIV-1 surface glycoprotein (gp120) is enhanced by the presence of the soluble CD4 receptor (McKeating, Cordell, Dean, Balfe, Virology 191, 732 -742, 1992). It has been shown in the present invention that HIV-1 entry is significantly blocked by both MAb 1F7 monoclonal antibody and IPI / KPR tripeptides whereas DPP IV peptidase activity was inhibited only by tripeptides. in question. MAb 1F7 antibody had no apparent effect on DPP IV activity (Tanaka et al.

  al, Proc. Natl. Acad. Sci. USA 90, 4586-4590, 1993).

  In addition, the same tripeptides did not affect the binding, whereas the MAb 1F7 monoclonal antibody 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 MAb 1F7 antibody affects the recognition site

of gp120 by the CD26 receptor.

  Material and method corresponding to the reported tests

Table 11 NIH 3T3 cells were transfected with plasmid vectors

  expressing CD4 and CD26 receptors, either independently or together, by the

  calcium phosphate coprecipitation technique.

  According to this technique, NIH 3T3 cells (1.2 106) were plated in 25 cm 2 flat vials 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 coprecipitation technique. After 48 hours, the transfected cells were washed with medium containing 2mM EDTA followed by further washing with the culture medium. The cells were then contacted with HIV1 LAI (corresponding to 0.5 gg of p25) for 6 hours in the absence or in the presence of HIV entry inhibitors (IPI, heparin). The cells were first washed in medium containing 2mM EDTA and then trypsinized to remove the extracellular virus. The cells were then respread in 75 cm2 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 EMC cells (5,106). The production of HIV1 (p25, ELISA) in EMC cell cultures was measured 7 and 11 days later in the supernatant. The CD4-expressing plasmid was 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 need for the presence of CD26 for entry of HIV into cells and the fact that cells producing HIV in in vitro cultures are doomed to death. 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 membrane level of infected cells, 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 cells, viral entry occurs only after cellular activation, a process associated with increased expression of the CD26 surface antigen. In addition, observations in HIV-infected individuals 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. and al, J. Immunol., 149, -3073-3077

  (1992); Vanham G. et al., J. AIDS 6, 749-757 (1993)).

  Such observations corroborate our results, demonstrating the need for CD26 for viral entry and the fact that cell cultures

  infected are sentenced to death.

  The gpl20 / gp41 complex of the HIV envelope plays a key role in the entry of the particles, and when this complex is expressed on the surface of the infected cells, it is responsible for the initiation of apoptosis by the 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 HIV entry mechanism described herein and 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

  induction of apoptosis by the gp120 / gp41 complex.

  The development of AIDS in seropositive individuals for HIV is correlated with the burden of virus 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 offers the means to develop new inhibitors of viral infection. The inhibition of the entry of HIV-1 and HIV-2 by IPI and KPR tripeptides gives the possibility of developing simple but specific inhibitors that could block the function of CD26 and therefore be used effectively as agents.

  therapeutic for AIDS patients.

51 2707170

TABLE N 1

SEQUENCES * V3 OF SOME ISOLATES

TYPICAL HIV-1, HIV-2 and SIV * 1_

  HIV-1 LAI CTRPNNNTRKSIRIQRGPGAFVTIGKIGNMRQAHC

  HIV-1 ELI CARPYQNTRQRTPIGLGQSLYTTRSRSIIGRAHC

  HIV-2 ROD CKRPGNKIVKQIMLMSGHVFHSHYQPINKRPRQAWC

  HIV-2 ISY CRRPENKTVVPITLMSGRRFHSQKIINKRPRQAWC

  SIV MM251 CRRPGNKTVLPVTIMSGLVFHSQPVNDRPKQAWC

  CPZ CHRPGNNTRGEVQIGPGMTFYNIENVVGDTRAYC

  SIV MND CHRKGHRSVVSTPSATGLLFYHGLEPGKNLKKGMC

  * The preserved patterns RP, KP and GPG are in bold type (Meyers et al. 1992)

TABLE N 2

  INHIBITION OF ENTRY OF HIV-1 LAI PARTICLES BY

PEPTIDES OF THE V3 * LOOP

  PEPTIDE RESIDUES INTERNALIZED p25% INHIBITION (a-a N) CELLS pg / 105 None - 168 0 gp120-IIIB 350-378 156 7 gp120-IIIB 418-441 138 18 gp41-IIIB 718-743 90 46

V3-IIIB 295-321 20 88

V3-1286 301-333 7 96

  V3-Summit LAI 307-327 62 63 V3-Summit HXB2 307-327 7 96

  * The sequences are described in Table 2a.

53 2707170

TABLE 2bis

  AMINO ACID SEQUENCES OF PEPTIDES USED

IN TABLE 2

  gpl20-IIIB (350-378) REQFGNNKTIIFKQSSGGDPEIVTHSFNC gpl20-IIIB (418-441) CRIKQIINMWQKVGKAMYAPPISG gp41-IIIB (718-743) QTHLPTPRGPDRPEGIEEEGGERDRD

  V3-IIIB (295-321) TRPNNNTRKSIRIQRGPGRAFVITGKIGNMRQAH

  V3-1286 (301-333) CTRPNNNTRKRIYLGPGRAWYTTKAIIGDIRQA

  V3-Summit LAI (307-327) CNTRKSIRIQRGPGRAFVTIGK V3-Summit HXB2 (307-327) CNTRKRIRIQRGPGRAFVTIGK

- 2707170

TABLE N 3

  INHIBITION OF THE ENTRY OF HIV-1 PARTICLES BY THE

TRIPEPTIDE IPI AND OTHER PEPTIDES

  PEPTIDE CONCENTRATION p25 INTERNALIZED% INHIBITION ICo (aM) CELLS pg / 105 mM

_- - 176 0

IPI 10 15 92 5

VPL 10 158 10

GPA 10 180 None

GPGG 10 98 73 8

GGG 10 185 None NV GP 10 170 None

KPR 10 91 6

RPGFSPEN 10 90 6

IPI O 190 O

IPI 1 82 57

IPI 5 45 76

IPI 10 15 92

TABLE No 4

  INHIBITION BY DIFFERENT PEPTIDES OF INFECTION

BY HIV-1 IN EMC CELLS

  TREATMENT PRODUCTION OF VIRUSES% INHIBITION

(p25 ng / ml) None 146 O

IPI 6 96

VPL 124 15

GPGG 45 69

GGG 138 6

  * measured in the culture medium at d + 4 post-infection.

56 2707170

TABLE 5

  INHIBITION OF PARTICLE ENTRY HIV-1 LAI

  MAbs SPECIFIC TO DPP IV / CD26 MAb: SPECIFICITY p25 INTRACELLULAR% INHIBITION CELLS pg / 105 None - 170 0

OKT4A CD4 12 93

OKT4 CD4 90 47

/ 4 Loop V3 25 85

BA5 DPP IV / CD26 62 64

1F7 DPP IV / CD26 19 89

  ALB1 CD10 175 No CDlla -LFA-1 180 No CD18 P-LFA-1 168 No

57 2707170

TABLE N 6

  INHIBITION OF DPP IV ACTIVITY ON CELLS

  EMC BY DIFFERENT PEPTIDES OR ANTIBODIES

  ADDITION O.D. 405 nm IC50 (M) None 0.215 IPI (20mM) 0.021 IPI (10mM) 3 GGG (20mM) 0.206

GGG (10M) NV

  MAb 1F7 0.240 MAb BA5 0.222 MAb ALB1 0.237

GPGG 6

KPR 5

RPGFSPFR 5

NV: invalid

* 58 2707170

TABLE N 7

  INHIBITION, BY IPI AND IF7 MONOCLONAL ANTIBODY

  SPECIFIC FOR DPPIV / CD26, INFECTION

BY HIV-2 EHO

  TREATMENT PRODUCTION OF VIRUSES% INHIBITION

RT cpm / ml None 182,640 0

IPI 40.220 78

  VPL 176,880 None MAb 1F7 33,780 82 MAb ALB1 180,460 None

  * measured in culture medium +4 post-infection.

TABLE 8

  HIV-1 INFECTION IS INHIBITED BY IPI TRIPEPTIDE

  BUT NOT BY OTHER INHIBITORS OF PROTEASES

  TARGET PROTEASE INHIBITOR PRODUCTION

VIRUS *

  p25 (ng / ml) No 176 IPI (10mM) DPP IV / CD26 18 Aprotinin Trypsin, Chymotrypsin Kallikrein, Plasmine 185 Leupeptin Trypsin, Plasmine Kallikrein, Cathepsin B 166 Papain Antipain, Trypsin Cathepsin B 223 Pepstatin Pepsin, Cathepsin D Renin 172 Bestatin Aminopeptidase B Leucine aminopeptidase 94 * in culture medium D + 4 post-infection

2707170

  rabia1 lisie - PIPtides V3 used in Mbibo studies: in life

HIV dar s 2es eelhluie CEM.

  These pepideids were obtained from P'ANRE. Inhibition of seizure of the virus has been

  In the above mentioned studies, the experiments described in Tables 3, 4 and 5 are discussed.

  At one: oncm-lration die 0.1-0.2 tIl 'e peptidC only pepftides SP92376 (HIV

  1286) and SP9363 (IV [b] B2) have involved the uptake of 90% of the virus control.

  5,9397 (HIV MN) resulted in an inhibition of all other peptides which showed no effect on the peptides.

  a.ynt an actvite, of inhibition sonrti e: i caractrts fat.

  E5: rz ::; 301-333 MNC-T-4-R - ## STR3 ## NOT -

T-I-R-Q-A

  S.t. 2, 23 7: 301.333 SF.2 C-T-R-1P.I'Si-N-T-R-I: 3-S - [- Y-1.G-P-G-R-A, .F-. i [T-T.G-R-I-I-G-

D-tq.-K-.A

  SP: 23: 3C, 13 '; FRZ: C-T-R-P-N-N-N-T-R-1-S-H-I-GPG-R-A-F-Y-A-T.G. -I-I-G-

D-I-R-Q-A

  S1P23'4: 301-333 4023 C-T-4R-PiIN-; ## STR5 ## wherein R 2 -N-R-1-C-β-C-β-F-Y-α-TG-D-1-pC [-R.Q.-SF 92375: 301-333 -067 C-T-R-P-nit-T-G-I-F Q-F'A-T-I]

D-I-.R - A

  SPC92 ',: 31-34' 1246 C-T-PR-P, -N-> - N-T '-: - R.i. [Y --- .-- G0-R-Y-W-K-T.T AI--

G-D-l-R-Q.What

  SI-P89;: 3021-324 LAI C-T-R-P-N..y: N-TRI-A-R-K-P-RS-G-4.Ip-I..T.AI.

D: ^ - IR-K-A, -I;

  SP904MZ: 32.3.3 CT-P-, N-T-R-LAI-I-P ---- KT-D-II

DMI-R-Q-a..c

  SFP323: 30I-323: C-TR-p-N'-N-T-R1-IR-T-K-G-PR-F-V-A-T-G-DI-I

  SPS936 6 M 37321 HfXB: a2 NTRK '------------- r .--

D-T-R-K-A

  SP93) 427 CRPN-NKR-iR G-RP-I-CQ-IY-TT-V-IG D) -I-R1-K.At SSM) 87: 307-32 IA T- -PN - TtIIRI-RP - AFVT

  SPF9708: 32S-, 33, T-R-P-N-N-1-t-K-5-1-I-R-C-W1 (P-C-A-V-TI.GK-I.G.

N-M-R-Q-.A.-H-C-N

  SP89369: 307-, 321 1A CNTRs: -: IRIQ- -GPGRAW-YVT-I-G, -K SP1937O ': 307-52: WXB2 CNTR-I-L-SR-iQC.PL ## STR2 ## ## STR3 ##, ## STR3 ##, ## STR2 ## ## STR2 ## ## STR1 ## ## STR2 ## ig3'7 CDC! [CETR: tV -. ^ GP3 ---- wPiw

  P893E70: 3D7F-32'7 WMJ] I -N - {-: R-I? .-, _-L - $ --.-, G-.P-GR-A-F -T-R-E3

  Table 10. The sensitivity of DPP IV activity in the presence of IPI peptide varies depending on whether the test

  is carried out on human or murine cells.

  Substrate Enzyme IC50 of IPI Human Murine GP-pNA peptidase DPP IV-like 50M 5mM RP-pNA peptidase DPP IV-like 50M 10mM R-pNA Arg-peptidase 10mM 10mM

63 2707170

  Table 11. Coexpression of human CD4 and CD26 receptors in NIH 3T3 murine cells makes them permissive

to HIV-1 infection.

  Transfection Inhibitor Day7 JourII (Plasmid) Production of HIV-1 (p25; μg / ml) (ng / ml)

None - 9-

CD4 - 8 5

CD26 - 11il 0.4

CD4 + CD26 - 206,200

CD4 + CD26 - 370 300

CD4 + CD26 IPI 20 2

CD4 + CD26 Heparin 8 -

Claims (21)

  1. Pharmaceutical composition, characterized in that it comprises as active ingredient, a compound containing a recognition pattern contained in a substrate of the DPPIV enzyme, or a pattern having a conformation sufficiently close to that of the aforementioned pattern of recognition. , to be recognized by the enzyme DPPIV and form with it an enzyme-substrate complex. 2. Pharmaceutical composition according to claim 1, characterized in that it comprises as active principle, a compound containing a recognition unit selected from the units GP, RP, KP, EP or DP, or from the GPG, RPG units. , KPG, EPG or DPG, the aforementioned motifs being either in the structural context of the peptide environment of the V3 loop of HIV or SIV envelope glycoproteins, or in a another context.   3. Pharmaceutical composition according to claim 1 or 2, characterized in that the recognition pattern is modified so as to preserve in vivo peptidase activities and in particular to prevent the enzyme cleavage activity normally exerted by the DPPIV on this ground.   4. Pharmaceutical composition according to claim 3, characterized in that the modification of the recognition pattern corresponds to a modification of the peptide bond preceding or following the Proline residue so that this bond is resistant to cleavage by the DPPIV.   5. Pharmaceutical composition characterized in that it comprises as active ingredient, a compound capable of modifying the interaction between on the one hand, a CD26 type receptor present on the cell surface of a patient infected with a retrovirus of the HIV type, in particular of the HIV-1 or HIV-2 type and, secondly, the glycoproteins of the envelope of this HIV retrovirus, this compound being associated with a heterologous or polymerized structure so as to favor its inhibition or prevention of   HIV or SIV retrovirus infection in vivo.   6. Pharmaceutical composition according to claim 5, characterized in that the active ingredient is included in a synthetic matrix of peptide type,   in particular to form a MAP type structure.   Pharmaceutical composition according to claim 5, characterized in that the compound constituting the   active ingredient is in polymerized form.   8. Pharmaceutical composition according to claim 1, characterized in that the compound constituting the active principle is in the form of a recombinant molecule, for example in the form of a protein   fusion with an antigen such as HBsAg.   9. Pharmaceutical composition according to any one   Claims 1 to 8, characterized in that the   active ingredient is a peptide containing a sequence   selected from GPGRAF, KRPGNK, RPGNK, KRPRQ or EPGKN.   10. Pharmaceutical composition according to any one   of claims 1 or 2, characterized in that the   ability of the compound to modify the interaction of the CD26 receptor vis-à-vis the glycoproteins of the envelope of the HIV retrovirus, is determined by the test comprising the following steps: - the contacting of a part of cells comprising at their surface the CD4 and CD26 human receptors, on the other hand in an amount equal to the TCID50, of an HIV human retrovirus, in particular -HIV-1 or HIV-2, or a simian SIV retrovirus, -the incubation said cells and HIV or SIV retrovirus at 37 C for a time sufficient to allow the retrovirus to enter the cells, in the presence of a determined amount of the test compound, - washing the cells to remove the retroviruses absorbed on the cells elimination of the extracellular virus and quantification of the intracellular virus; centrifugation to separate the retroviral proteins, for example at 1000 g, and recovery of the supernatant,   - the detection of HIV proteins.   11. Compound capable of inhibiting in vivo the infection with an HIV human retrovirus, in particular a retrovirus of the HIV-1 or HIV-2 type, characterized in that it is a compound capable of modifying or to inhibit the interaction between, on the one hand, a CD26 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 a nature peptide or nonpeptide, this compound containing a recognition pattern contained in a substrate of the DPPIV enzyme, or a pattern having a conformation sufficiently close to that of the aforesaid recognition motif, to be recognized by the DPPIV enzyme and form with it an enzyme-substrate complex. 12. Compound according to claim 11, characterized in that the recognition pattern meets the definition given in any of the Claims 2 to 9.   13. Compound according to any one of the claims   11 or 12, characterized in that it is either associated with a heterologous structure for example to form a MAP-type structure or a recombinant protein, be polymerized.   14. Recombinant cell, characterized in that it is capable of expressing CD4 receptors together and human CD26.   15. Recombinant cell according to claim 14, characterized in that it is a eukaryotic cell, in particular a human cell, for example a HeLa cell or a murine cell, for example an NIH 3T3 cell.   16. Transgenic animal characterized in that its somatic cells and / or its germinal cells comprise a sequence coding for the human CD4 receptor and a coding sequence for the human CD26 receptor, under conditions allowing expression joint of these two receivers.   An animal according to claim 16 as obtained by introducing into the cells of the animal at a stage of early embryonic development, e.g. not exceeding 8 cells, a coding sequence for the human CD4 receptor and a coding sequence for the human CD26 receptor, under conditions allowing   the joint expression of these two receptors.   18. Transgenic animal characterized in that it is a descendant of a transgenic animal according to the   claim 16 or claim 17.   19. Transgenic animal according to any of   claims 16 to 18, characterized in that it is   of a mammal, for example a rodent for example a mouse or a monkey.   20. A method for obtaining a transgenic animal comprising in its somatic cells and / or in its germinal cells a coding sequence for the human CD4 receptor and a coding sequence for the human CD26 receptor, under conditions allowing joint expression. of these two receptors comprising the steps of: introducing a coding sequence for the human CD4 receptor, for example a genomic sequence or a cDNA sequence, into the cells of the animal, at an early stage of development embryo, for example not exceeding 8 cells, introducing into another animal of the same species, 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 an early stage of embryonic development, for example not exceeding 8 cells, - the crossing of animals resulting from the development of e mbryons thus modified, under conditions which make it possible to obtain a transgenic animal which expresses, in its somatic cells and / or in its germ cells, CD4 and CD26 human receptors.   21. Vaccine for protection against infection by an HIV or SIV retrovirus, characterized in that it comprises the active ingredient as defined in one of the   any of claims 1 to 9 or the compound according to   any one of claims 11 to 13, in   conditions allowing the production of antibodies   neutralizers against HIV or SIV infection.