WO2006027468A2

WO2006027468A2 - Hla-dp4 restricted t cd4+ du vih epitopes and the use thereof

Info

Publication number: WO2006027468A2
Application number: PCT/FR2005/002152
Authority: WO
Inventors: William Cohen; Sandra Pouvelle-Moratille; Gaétan MUNIER; Bernard Maillere
Original assignee: Commissariat A L'energie Atomique
Priority date: 2004-08-27
Filing date: 2005-08-26
Publication date: 2006-03-16
Also published as: WO2006027468A3; FR2874612A1

Abstract

The invention relates to antigenic peptides of a human immunodeficiency virus (VIH) comprising at least one type of T CD4+ epitope which is restricted to a HLA-DP4 molecule and to the vaccine and diagnosis use thereof.

Description

HLA-DP4-RESTRICTED HIV CD4 + EPITOPES AND USES THEREOF

The present invention relates to antigenic peptides of the human immunodeficiency virus (HIV) comprising at least one CD4 + T epitope which is restricted to an HLA-DP4 molecule, and to their vaccine and diagnostic applications.

CD4 + T cells play an essential role in controlling HIV infection as evidenced by the high frequency of HIV-specific CD4 + T cells in non-progressing individuals who have been infected for more than 12 years and do not develop disease (AIDS) in the absence of any antiviral treatment; in contrast, low or undetectable levels of HIV-specific CD4 + T cells are observed in the majority of individuals with recent seroconversion or chronic HIV infection who are untreated.

Indeed, CD4 + T lymphocytes play a major role in the induction and maturation of humoral and cellular immunity. Via cellular contacts and the secretion of many cytokines, they induce the activation of antigen presenting cells (APCs for Antigen Presenting CeIl) which in turn recruit HIV-specific CD8 + T cells. They are also involved in the maturation of effector cells that are cytotoxic T lymphocytes. Finally, they can themselves play the role of effectors by producing anti-viral lymphokines such as IFN-γ and TNF-α. Thus, a decrease in the level of CD4 + T cells that reduces the effectiveness of the immune system is a major cause of progression to the disease during HIV infection.

The activation of CD4 + T lymphocytes is due to the presence of antigenic peptides by the major histocompatibility complex type II molecules carried by the antigen presenting cells; in humans, they are called HLA II molecules for Human Leucocyte Antigen type IL These antigenic peptides, called T epitopes, result from the proteolytic degradation of the antigens by the antigen presenting cells. They have variable lengths, generally from 13 to 25 amino acids and have a sequence that makes them capable of binding to HLA II molecules. It is well known that, like the native antigen, a peptide comprising a CD4 + T epitope is capable of to stimulate specific CD4 + T lymphocytes in vitro or to recruit them in vivo. It is therefore sufficient to induce a CD4 + T response.

These observations are in favor of the use of such HIV-specific antigenic peptides capable of stimulating a strong CD4 + T response for the preparation of an anti-HIV vaccine composition in humans, especially in combination with other epitopes. B or T CD8 + HIV.

Indeed, even if considerable progress in the field of chemotherapy makes it possible to hope for the restoration of normal immunity in patients infected with the virus, the induction of a protective antiviral response requires, in the majority of cases, specific treatment such as a vaccine.

Such peptides that are recognized by HIV-specific CD4 + T cells are also useful as reagents in a diagnostic test of infection or treatment follow-up in humans, based on direct detection (lymphocyte proliferation assay) or indirect (production of antibodies, cytokines, etc.) of said CD4 + T lymphocytes.

However, one of the major problems which limits the use of these peptides as antigen is the identification of CD4 + T epitopes, since their sequence varies from one individual to another due to the polymorphism of HLA IL molecules. the HLA II molecules are heterodimers consisting of a polymorphic alpha (α) chain and a beta (β) chain. There are four types of HLA II molecules per individual (2 HLA-DR, 1 HLA-DQ and 1 HLA-DP), named according to the allele encoding the beta chain which is the most polymorphic. The HLA-DR molecule is very polymorphic. Indeed, while its alpha chain has only 3 alleles, the beta (β) chain encoded by the DRB1 gene, which is the most expressed, currently has 458 alleles. For the HLA-DQ and HLA-DP molecules, the two chains (α and β) that constitute them are polymorphic but they have fewer alleles. There were 8 DQAl alleles (HLA-DQ α chain), 56 DQB1 alleles (HLA-DQ β chain), 20 DPA1 alleles (HLA-DP α chain), and 110 DPBl alleles (HLA-DP β chain). . However, the combination of the two α and β chains encoded by these alleles gives rise to numerous HLA-DQ and HLA-DP molecules. Because of this poly¬ morphism, these isoforms possess different binding properties to each other, which implies that they can bind different peptides of the same antigen. Thus, each individual recognizes in an antigen a set of peptides whose nature depends on the HLA II molecules that characterize it. Since there are a large number of HLA II alleles, there is therefore in a given sequence an important repertoire of T epitopes of very different sequences, each specific for a different allele.

Thus, a peptide capable of stimulating an HIV-specific CD4 + T response in some individuals may be inactive in the majority of other individuals because they do not recognize HIV proteins by the same epitopes. Most of the HIV CD4 + T epitopes already described are localized in Gag, Env, PoI and Nef proteins (Gag: Wilson et al., J. Virol., 2001, 75, 4195-4207, Malhotra et al., J. Clin., Invest, 2001, 107, 505-517, Venturini et al., J. Virol., 2002, 76, 6987-6999, Boritz et al., J. Immunol., 2003, 170, 1107-1116, Kaufmann et al. al., J. Virol., 2004, 78, 4463-4477; International Applications WO 98/32456 and WO 01/24810; Eny: Malhotra et al., J. Virol., 2003, 77, 2663-2674; Lekutis et al. J. Immunol., 1997, 159, 2049-2057, International Applications WO 98/32456 and WO 01/24810, PoI: Manca et al., J. Acquir, Immun Defic., Hum Syndrome, Retrovirol. 9, 227-237, Van der Burg et al., J. Immunol., 1999, 162, 152-160; International Applications WO 98/32456 and WO 01/24810; Nef: Wentworth et al, Vaccine, 1994, 12, Kaufmann et al., International Applications WO 02/062834 and WO 01/24810).

All these CD4 + T epitopes are restricted to one HLA DR or DQ molecule, excluding that carried by the peptide Nef 180-193 which is restricted to HLA-DP5, a non-dominant molecule in the Caucasian population (allele frequency of 1.3%). For the HLA-DR molecules, a dozen alleles are necessary to cover more than 60% of the gene frequency found in the Caucasian population and therefore affect more than 85% of this population (International Application WO 02/062834). Thus, a peptide carrying a HLA-DR restricted epitope is generally not recognized by all the predominant HLA-DR molecules in the population (see, in particular, Table 4 of Kaufmann et al., Supra, and Table VII. of International Application WO 02/062834). Accordingly, each peptide carrying an HLA-DR restricted epitope must be tested against a tens of HLA-DR molecule to select the peptides recognized by the majority of these molecules, and it is generally necessary to use a mixture of at least two or three different peptides to obtain an effective vaccine in the majo¬ rity individuals from the population to be vaccinated. The inventors have identified other CD4 + T epitopes derived from the HIV PoI (RT), Env, Vpr and Vif proteins, which are restricted to an HLA-DP4 molecule and are capable of inducing, by themselves, a CD4 + T response. specificity of HIV, in the majority of vaccinated individuals. Indeed, HLA-DP4 molecules are very common, especially in the Caucasian population. Thus, two DP4 molecules (DP401 and DP402) alone cover the majority of the HLA-DP allelic frequency in the Caucasian population (around 50% in Europe and 80% in North America) and are also present at non-negligible frequencies in the other populations (allelic frequency of the order of 60% in South America, 60% in India, 25% in Africa and 15% in Japan). Each of these DP4 molecules comprises an alpha chain coded, either by Pallele DPAl * 0103 which is the most frequent (78.2%), or by the DPAl * 0201 allele (20.2%) and a beta chain encoded by allele DPB1 * 0401 (molecule HLA-DP401) or DPB1 * 0402 (molecule HLA-DP402).

The subject of the present invention is therefore an HIV peptide comprising a CD4 + T epitope capable of being presented by an HLA-DP4 molecule and inducing HIV-specific CD4 + T cells, selected from the group consisting of: a) fragments of 13 consecutive amino acids of the Env, RT, Vpr and Vif proteins located between the following positions:

339 to 351, 264 to 276, 347 to 359, 404 to 416, and 409 to 421 of the RT protein,

32 to 44, 389 to 401, 484 to 496, 621 to 633, 671 to 683, 678 to 690, 685 to 697, 750 to 762 and 761 to 773 of the Env protein,

16 to 28 and 62 to 74 of the Vpr protein, and

- 62 to 74 of the Vif protein, b) fragments of 14 or 15 consecutive amino acids of said RT, Env, Vif and Vpr proteins, which include the positions defined in a), and in particular peptides RT 338-352, 263- 277, 346-360, 403-417, 408-422; Ca. 31-45, 388-402, 483-497, 620-634, 670-684, 677-691, 684-698, 749-763 and 760-774, Vpr 15-29, 61-75 and Vif 61-75, and c) the variants of the defined peptides in a) or b), which have an identical length and a binding activity to an HLA-DP4 molecule less than or equal to 100O nM, for use as an antigen in prophylactic or therapeutic vaccination, or the diagnosis of HIV infection.

The peptides according to the invention have the following properties:

HLA-DP4 binding activity: the peptides according to the invention have a high affinity for HLA-DP4 (binding activity <1000 nM); the binding activity can be measured by an HLA-DP4 / peptide binding test, in competition, with immunoenzymatic revelation, according to the principle described in PCT International Application WO 03/040299,

immunogenicity: the peptides according to the invention are recognized by CD4 + T lymphocytes in the majority of individuals because of the preponderance of the HLA-DP4 molecule in the Caucasian population but also in populations of South America and India, and to a lesser extent those in Africa and Japan. These peptides are also capable of efficiently inducing HIV-specific CD4 + T cells from the precursors present in the majority of naive or infected individuals, or to stimulate such HIV-specific lymphocytes present in the majority of infected individuals. In addition, the CD4 + T lymphocytes induced by these peptides recognize the epitope of the native antigen naturally presented by the dendritic cells as well as natural variants of said peptides. Accordingly, such peptides represent potential candidates for prophylactic or therapeutic vaccination against HIV infection, particularly of interest because they can induce an HIV-specific CD4 + T response in the majority of individuals in the population. In addition, peptides whose sequence is conserved in natural variants of HIV or which cross-react with CD4 + T epitopes recognized by natural variants of HIV are capable of inducing a CD4 + T response to variants of HIV. The immunogenicity of the peptides can be determined, in particular from peripheral blood mononuclear cells (PBMCs), by any appropriate assay. Preferred is known to those skilled in the art such as, for example, a cell proliferation assay, an ELISPOT assay (cytokine-producing cell assay) or an intracellular cytokine assay (IFN-gamma, IL-2, IL-2). 4 and IL-10).

The invention encompasses peptides whose sequence corresponds to that of a fragment of the Env, RT, Vpr and Vif protein of any HIV isolate (HIV-1 or HIV-2) including natural variants of HIV, isolated from individuals infected with HIV. The HIV-I isolates are classified into three groups (M, N (non-M, non-O) and O) which are divided into subtypes or clades according to their genetic similarity (Env gene); group M comprises at least 9 subtypes (subtypes A to I), subtype B being predominant in western countries. The genome sequences and corresponding proteins of the different HIV isolates are available in the databases, including NCBI ( ^" http://www.ncbi.nlm.nih.gov ^" ) or LANL (Los Alamos National Laboratory). http: //www.hiv.lanl.govy NCBI access numbers K03455 and AF075719 include, respectively, the HIV-I group M subtype B subunit sequence, designated HXB2, corresponding to the LAI isolate. , and the sequence of isolate MN In addition, consensus sequences defined from natural variants of different HIV-I isolates are also available in the databanks, especially for HIV group M subtype B -I (http://hiv.lanl.gov/content/hiv-db/CONSENSUS/M GROUP / Consensus.html).

The positions of the peptides derived from RT, Vif, Vpr or Env proteins are indicated relative to the HXB2 reference sequence. From these indica¬ tions, one skilled in the art is able to easily determine the corresponding positions in the proteins of other HIV isolates, which may vary from a few amino acids due to short insertions and / or deletions in the amino acid sequence of said proteins.

The invention also encompasses variants obtained from the preceding sequences by mutation (insertion, deletion, substitution) of one or more amino acids in the peptide sequence, since said variant retains a high affinity for HLA-DP4 (activity <1000 nM) and is immunogenic. The amino acid residues involved in the binding to HLA-DP4 (anchor residues P1, P4, P6 and P9) and the effect of the modifications of these residues on the binding to HLA-DP4 are known to those skilled in the art; PCT International Application WO 03/040299 teaches in particular that the residues at P6, P1 and / or P9 must be almost exclusively aromatic or hydrophobic whereas the residue at P4 may be any amino acid residue. Among these variants, mention may be made of those in which the anchoring residues P6, P1 and / or P9 are unchanged, and at least one of the other amino acid residues is modified. These variants are in particular the peptides comprising the sequence of 9 consecutive amino acids of the proteins Env, RT, Vpr and Vif including the anchoring residues P1, P4, P6 and P9, which sequence corresponds to residues located at positions +3 to +11 , relative to the sequence of peptides of 13 amino acids as defined above. These are peptides of 13 to 15 amino acids including at least the sequence which is located between the following positions: 34 to 42, 391 to 399, 486 to 494, 623 to 631, 673 to 681, 680 to 688, 687 at 695, 752 to 760 and 763 to 771 of the Env protein; 341 to 349, 159-167, 266 to 274, 349 to 357, 406 to 414, and 411 to 419 of the RT protein; 18 to 26 and 64 to 72 of the Vpr protein; 64-72 of the Vif protein.

The invention also encompasses modified peptides derived from the foregoing by introduction of any modification at amino acid residue (s), peptide bond or peptide ends, as long as said modified peptide retains a high affinity for HLA. -DP4 (binding activity <1000 nM) and is immunogenic. These modifications, which are introduced into the peptides by conventional methods known to those skilled in the art, include, without limitation: the substitution of an amino acid by a non-proteinogenic amino acid (amino acid D or the like of an acid amine); adding a chemical group (lipid, oligo or polysaccharide) at a reactive function, in particular the side chain R; modification of the peptide bond (-CO-NH-), in particular by a retro-type or retro-inverso bond (-NH-CO-) or a bond different from the peptide bond; cyclization; the fusion of a peptide (epitope of interest for vaccination, label useful for the purification of the peptide, especially in cleavable form with a protease); the fusion of the sequence of said peptide with that of a protein, in particular an α or β chain of an HLA-DP4 molecule or the extracellular domain of said chain; coupling to a suitable molecule, in particular a marker, for example a fluorochrome. These modifications are intended in particular bind to increase the stability, solubility, immunogenicity or to facilitate the purification or detection of either the peptide according to the invention or CD4 + cells specific for said peptide.

According to an advantageous embodiment of said peptide, it is selected from the group consisting of peptides RT 338-352, 339-351, 346-360, 347-361, 403-417, 404-416, Env 31-45 , 32-44, 388-402, 389-401, 483-497, 484-496, 620-634, 621-633, 677-691, 678-690, 684-698, 685-697 and Vpr 15-29, 16-28, 61-75 and 62-74. Preferably, said peptide is selected from the group consisting of peptides RT 338-352, 339-351, Env 31-45, 32-44, 388-402, 389- 401, 620-634, 621-63 and Vpr 15-29, 16-28, 61-75 and 62-74. Preferably, said peptide is selected from the group consisting of peptides RT 338-352, Env 31-45, 388-402, and 620-634. Even more preferably, said peptide is selected from the group consisting of peptides RT 338-352 and Env 31-45.

According to another advantageous embodiment of said peptide, its sequence corresponds to that of a type 1 HIV isolate, preferably of the M group, preferably said sequence corresponds to the reference sequence of the subtype B, called HXB2. (NCBI access number K03455).

According to an advantageous arrangement of this embodiment, said peptide is selected from the group consisting of the sequences SEQ ID NO: 2 to 7, 16 to 20, 22, 24 to 29, 38 to 42, 44 to 48 and 50 to 56 .

According to another advantageous embodiment of said peptide, it is labeled or complexed; it can in particular be complexed with labeled (biotinylated) HLA-DP4 molecules to form HLA-DP4 / peptide complexes, in particular multimeric complexes such as tetramers. According to yet another advantageous embodiment of said peptide, said DP4 molecule comprises a β chain encoded by the DPB1 * 0401 allele (HLA-DP401 molecule) or DPB1 * 0402 (HLA-DP402 molecule); the α chain being encoded by the DPAl * 0103 or DPAl * 0201 allele.

The present invention also relates to a polyepitopic fragment comprising the concatenation of at least two identical or different epitopes, at least one of which is a peptide as defined above, for a antigen in prophylactic or therapeutic vaccination, or the diagnosis of HIV infection.

Preferably, said polyepitopic fragment has a length of 20 to 1000 amino acids, preferably 20 to 100 amino acids. Said polyepitopic fragment advantageously comprises a label fused at one of its ends for the purification or detection of said fragment. The tag, especially a polyhistidine sequence or a B epitope of another virus, is preferably separated from the polyepitope sequence by a protease cleavage site so as to isolate the polyepitopic sequence from the fusion.

According to an advantageous embodiment of said polyepi¬ topical fragment, it comprises the concatenation of at least one CD4 + T epitope as defined above and an epitope selected from the group consisting of:

a CD8 + T epitope of an HIV protein presented by an HLA I molecule and recognized by HIV-specific cytotoxic T lymphocytes; HIV CD8 + T epitope sequences are available from the LANL database (http://www.hiv.lanl.gov/content/immunologv/maps/ctl/ctl.pdf),

a CD4 + T epitope of the HIV Nef protein, in particular an epitope binding to at least one predominant HLA DR molecule in the Caucasian population, as defined in PCT International Application WO 02/062834 (Nef 1-36, 66- 94, 74-88, 77-91, 137-168, 139-153, 175-190, 182-198, 178-192, 181-195, 183-197, 187-201 and 189-203),

- a CD4 + T epitope universal natural or synthetic such as the peptide of the tetanus toxin TT 830-846 (.. O ¹ SULLIVAN et al, J. Immunol, 1991, 147, 2663-2669), the haemagglutinin peptide virus HA 307-319 (O'SULLIVAN et al, supra), PADRE peptide (KXV AA WTLKAA, Alexander et al., Immunity, 1994, 1, 751-761), peptide 45-69 of HIV Nef protein And peptides derived from Plasmodium falciparum antigens such as the CS.T3 peptide (SINIGAGLIA et al., Nature, 1988, 336, 778-780) and the CSP, SSP2, LSA-I and EXP-I peptides (DOOLAN et al., J. Immunol., 2000, 165, 1123-1137),

an epitope B constituted by a sugar (ALEXANDER et al., cited above), said epitope B preferably being in the form of a glycopeptide, and an epitope B of an HIV protein specifically recognized by antibodies directed against HIV; Sequences of HIV B epitopes are available in the LANL database (http://www.hiv.lanl.gov/content/imrnunology/rnaps/ab/ab.pdf). The combination of the CD4 + T epitope with at least one of the epitopes as defined above advantageously makes it possible to trigger or modulate an anti-HIV immune response.

The present invention also relates to a lipopeptide comprising a peptide or a polyepitopic fragment as defined above, for use as an antigen in prophylactic and therapeutic vaccination or the diagnosis of HIV infection.

Said lipopeptide is especially obtained by adding a lipid on an α-amino function or on a reactive function of the side chain of an amino acid of said peptide or polyepitopic fragment; it may comprise one or more chains derived from C _4-20 fatty acids, optionally branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-amino hexadecanoic acid, pimelautide, trimetauxide) or a derivative thereof of a steroid. The preferred lipid portion is in particular represented by a N ^α -acetyl-lysine N ^ε (palmitoyl) group, also called Ac-K (Pam). The present invention also relates to a fusion protein consisting of a protein or a fragment of protein, fused with a peptide or a polyepitopic fragment as defined above, for use as an antigen in prophylactic and therapeutic vaccination or the diagnosis of HIV infection. The peptide or polyepitopic fragment may be fused with the NH ₂ or COOH end of said protein or inserted into the sequence of said protein.

Advantageously, said fusion protein consists of an HIV peptide as defined above, fused with one of the chains of an HLA-DP4 molecule, preferably the beta chain, or with a fragment thereof. to a soluble HLA-DP4 molecule, in particular a fragment corres¬ coping with the extracellular domain preceded by the homologous signal peptide or a heterologous signal peptide. Said HIV peptide is advantageously inserted between the signal peptide and the NH ₂ end of the extracellular domain of the β chain, as described for the HLA-DR molecule (Kolzin et al., PNAS, 2000, 97, 291-296).

Alternatively, said HIV peptide or said polyepitic fragment are fused with a protein facilitating their purification or detection, known to those skilled in the art such as in particular Glutathione-S-Transferase (GST) and fluorescent proteins (GFP and derivatives ). In this case, the sequence of the peptide or polyepitopic fragment of interest is preferably separated from the rest of the protein by a protease cleavage site, to facilitate the purification of said peptide or said polyepitopic fragment.

The subject of the present invention is also an expression vector comprising a polynucleotide encoding a peptide, a polyepitopic fragment or a fusion protein as defined above, for use as an antigen in the prophylactic or therapeutic vaccination of the subject. HIV infection.

According to the invention, the sequence of said polynucleotide is that of the cDNA encoding said peptide or polyepitopic fragment or said fusion protein. Said sequence may advantageously be modified in such a way that codon usage is optimal in the host in which it is expressed. In addition, said polynucleotide may be linked to at least one heterologous sequence.

Within the meaning of the present invention, hetero¬logical sequence is meant in relation to a nucleic acid sequence encoding an HIV peptide, any nucleic acid sequence other than those which, in nature, are immediately adjacent to said sequence of nucleic acid sequences. nucleic acid encoding said HIV peptide. According to the invention said recombinant vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate transcriptional regulatory and possibly translational sequences (promoter, activator, intron, codon of (ATG), stop codon, polyadenylation signal). Many vectors in which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known. in themselves; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or integration into the chromosomal material of the host), as well as the nature of the host cell. For example, it is possible to use, inter alia, viral vectors such as adenoviruses, retroviruses, lentiviruses, AAVs and baculoviruses, in which the sequence of interest has been inserted beforehand; said sequence (isolated or inserted in a plasmid vector) may also be associated with a substance enabling it to cross the membrane of the host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or the to introduce into said host cell using physical methods such as electroporation or microinjection. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes.

The subject of the present invention is also an immunogenic or vaccinal composition, characterized in that it comprises at least one HIV peptide, a polyepitopic fragment, a lipopeptide or a vector as defined above, and a pharmaceutically acceptable vehicle, and or a carrier substance, and / or an adjuvant.

The immunogenic composition according to the invention is in a galenic form suitable for parenteral (subcutaneous, intramuscular, intravenous), enteral (oral, sublingual), or local (rectal, vaginal) administration.

The pharmaceutically acceptable vehicles, the carrier substances and the adjuvants are those conventionally used. The adjuvants are advantageously chosen from the group consisting of: oily emulsions, mineral substances, bacterial extracts, saponin, alumina hydroxide, monophosphoryl lipid A and squalene.

The carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, solid microspheres of saccharide nature (poly (lactide-co-glycolide)) or auriferous, and nanoparticles. Preferably, said immunogenic composition comprises at least one HIV peptide including an HLA-DP4 restricted CD4 + T epitope as defined above and another HIV peptide including a CD8 + T epitope, in the form of a mixture of peptides, a polyepitopic fragment or an expression vector encoding said peptides or said fragment.

Preferably, said composition also comprises at least one other peptide including an epitope selected from the group consisting of: a CD4 + T epitope of the HIV Nef protein, a universal CD4 + T epitope, a B epitope consisting of a sugar and a HIV B epitope as defined above. The present invention also relates to the use of a peptide, a polyepitopic fragment, a lipopeptide or a vector as defined above, for the preparation of a vaccine intended for the prevention and / or or the treatment of HIV infection in an HLA-DP4 positive individual.

The subject of the present invention is also the use of a peptide as defined above, for the preparation of a reagent for diagnosing the immune status of a HLA-DP4 positive individual, with respect to the HIV.

For the purposes of the present invention, the term "diagnosis of the immune status of an individual with respect to HIV" means the detection of the presence in said individual of CD4 + T cells specific for an HIV antigen. This detection makes it possible to monitor the evolution of the HIV-specific CD4 + T response during natural infection or antiviral treatment or HIV vaccination.

The detection is carried out from a biological sample containing CD4 + T lymphocytes, in particular a sample of mononuclear cells isolated from a peripheral blood sample (PBMCs). The present invention also relates to a method for diagnosing the immune status of an individual with respect to HIV, characterized in that it comprises:

contacting a biological sample of said individual with a peptide as defined above, and detecting antigen-specific CD4 + T lymphocytes.

HIV (RT, Vif, Vpr or Env), by any appropriate means. The subject of the present invention is also a kit for detecting the immune status of an individual with respect to HIV, characterized in that it comprises at least one peptide as defined above, associated with a means for detection of CD4 + T lymphocyte cells specific for an HIV antigen. The detection of antigen-specific CD4 + T lymphocytes is carried out by any means, known in themselves. For example, it is possible to use direct means such as flow cytometry in the presence of multimeric complexes, or indirect means such as lymphocyte proliferation assays and cytokine assays such as IL-2, IL-4, IL-5, IL-10 and IFN-gamma, in particular by immunoenzymatic techniques (ELISA, RIA, ELISPOT) or by flow cytometry (intracellular cytokine assay). More precisely:

A cell suspension (PBMC, PBMC depleted in CD8 + cells, T cells previously enriched by an in vitro culture step with the peptides as defined above or cloned T lymphocytes) is cultured for 3 to 5 days in the presence of said peptides. and if necessary appropriate screening cells, such as dendritic cells, autologous or heterologous PBMCs, lymphoblastoid cells such as those obtained after infection with EBV or genetically modified cells. The presence of HIV-specific CD4 + T cells in the initial suspension is detected using the HIV peptides, using one of the following methods: * Proliferation test:

The proliferation of HIV-specific CD4 + T cells is measured by incorporation of tritiated thymidine into the DNA of the cells. * ELISPOT test:

The ELISPOT test makes it possible to reveal the presence of secretory T cells of cytokines (IL-2, IL-4, IL-5, IL-10 and IFN-gamma), specific for a peptide such as as defined above. The principle of this test is described in Czerkinsky et al., J. Immunol. Methods, 1983, 65, 109-121 and Schmittel et al, J. Immunol. Methods, 1997, 210, 167-174, and its implementation is illustrated in International Application WO 99/51630 or Gahéry-Ségard et al., J. Virol., 2000, 74, 1694-1703. * detection of cytokines:

The presence of HIV-specific T cells secreting cytokines such as IL-2, IL-4, IL-5, IL-10 and IFN-γ is detected, either by assaying the cytokines present. in the culture supernatant, by an immunoenzymatic test, in particular using a commercial kit, or by detection of intracellular cytokines in flow cytometry. The principle of detection of intracellular cytokines is described in Goulder et al. , J. Exp. Med., 2000, 192, 1819-1832 and Maecker et al., J. Immunol. Methods, 2001, 255, 27-40 and its implementation is illustrated in Draenert et al., J. Immunol. Methods, 2003, 275, 19-29. * multimeric complexes.

a biological sample, preferably peripheral blood mononuclear cells (PBMC), is brought into contact with marked multimeric complexes, in particular with a fluorochrome, formed by the binding between soluble HLA-DP4 molecules and HIV peptides, such as defined above, and the cells labeled with said multimeric complexes are analyzed, in particular by flow cytometry.

Advantageously, prior to contacting the biological sample with said complexes, it is enriched in CD4 + T lymphocytes, bringing it into contact with anti-CD4 antibodies. The HLA-DP4 / peptide multimeric complexes can be prepared from native molecules extracted from cells expressing HLA-DP4 or from recombinant molecules produced in appropriate host cells as specified, for example in NOVAK et al. (J. Clin. Investig., 1999, 104, R63-R67) or in KURODA et al. (J. Viral., 2000, 74, 18, 8751-8756). These HLA-DP4 molecules can in particular be truncated (deletion of the transmembrane domain) and their sequence can be modified in order to make them soluble or to facilitate the pairing of alpha and beta chains (Novak et al., Cited above).

The loading of HLA-DP4 molecules with the peptide can be done by contacting a preparation of HLA-DP4 molecules as above, with the peptide. For example, soluble and biotinylated HLA-DP4 molecules are incubated, for 72 hours at 37 ^° C., with a 10-fold excess of HIV peptides as defined above, in a 10 mM phosphate-citrate buffer, NaCl 0.15M to a pH of between 4.5 and 7.

Alternatively, the peptide sequence can be introduced into one of the chains of the DP4 molecule in the form of a fusion protein which allows the preparation of HLA-DP4 / peptide multimeric complexes from appropriate host cells expressing said protein of fusion. Said complexes can then be labeled, in particular with biotin.

The multimeric complexes of tetramer type are obtained in particular by adding to the HLA-DP4 loaded molecules, streptavidin labeled with a fluorochrome in a quantity four times lower (mole to mole) relative to HLA-DP4 molecules. The assembly is then incubated for a sufficient time, for example overnight at room temperature.

The multimeric complexes can also be formed, either by incubation of HLA-DP4 monomers / peptides with streptavidin-coupled magnetic beads as described for the HLA I molecules (Bodinier et al., Nature, 2000, 6, 707-710), or by inserting HLA-DP4 / peptide monomers into lipid vesicles as described for murine class II MHC molecules (Prakken, Nature Medicine, 2000, 6, 1406-1410).

In order to use these multimeric HLA-DP4 / peptide complexes, in particular of the tetramer type, a suspension of cells (PBMC, PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with HIV peptides such as defined above or cloned T lymphocytes) with multimeric HLA-DP4 / peptide complexes at a suitable concentration (for example of the order of 10 to 20 μg / ml), for a time sufficient to allow the binding between the complexes. and HIV-specific CD4 + T cells (e.g., on the order of 1 to 3 hours). After washing, the suspension is analyzed by flow cytometry: the labeling of the cells is visualized by the multimeric complexes which are fluorescent.

Flow cytometry makes it possible to separate the cells labeled with the HLA-DP4 / peptide multimeric complexes from the unlabeled cells and thus perform cell sorting.

The present invention thus also relates to a method for sorting CD4 + T lymphocytes specific for an HIV antigen, characterized in that includes at least the following steps:

contacting a cell sample with labeled HLA-DP4 / peptide multimeric complexes, in particular with a fluorochrome, said complexes being formed by the binding of soluble HLA-DP4 molecules with at least one HIV peptide as defined herein; above, and

sorting the cells bound to said HLA-DP4 / peptide complexes, in particular in flow cytometry.

The present invention further relates to a peptide as defined above, excluding peptides RT 338-352, 403-417, Env 483-497, 684-698 and Vpr 15-29.

The present invention further relates to a polyepitopic fragment, a lipopeptide, a polynucleotide, an expression cassette, a recombinant vector derived from the preceding peptides.

The invention encompasses in particular: a) expression cassettes comprising at least one polynucleotide as defined above, under the control of appropriate transcriptional regulatory and optionally translational sequences (promoter, activator, intron, initiation codon (ATG), stop codon, polyadenylation signal), and b) recombinant vectors comprising a polynucleotide according to the invention. Advantageously, these vectors are expression vectors comprising at least one expression cassette as defined above.

The polynucleotides, the recombinant vectors and the trans¬ formed cells as defined above are particularly useful for producing the peptides, polyepitopic fragments and fusion proteins according to the invention. The polynucleotides according to the invention are obtained by conventional methods, known per se, according to the standard protocols such as those described in Current Protocols in Molecular Biology (Frederick A. USUBEL, 2000, Wiley and Son Inc., Library of Congress, USA). For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or by total or partial chemical synthesis. The recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.

Peptides and their derivatives (variants, modified peptides, lipopeptides, polyepitopic fragments, fusion proteins) as defined above, are prepared by standard techniques known to those skilled in the art, in particular by solid phase synthesis or liquid or by expression of a recombinant DNA in a suitable cellular system (eukaryotic or prokaryotic).

More precisely,

the peptides and their derivatives (variants, polyepitopic fragments) can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am Chem Soc., 1964, 85: 2149-) and purified by reverse phase high performance liquid chromatography,

the lipopeptides may especially be prepared according to the process described in International Applications WO 99/40113 or WO 99/51630. the peptides and derivatives such as the variants, the poly¬ epitopic fragments and the fusion proteins can also be produced from the corresponding cDNAs, obtained by any means known to those skilled in the art; the cDNA is cloned into a eukaryotic or prokaryotic expression vector and the protein or fragment produced in the cells modified with the recombinant vector are purified by any appropriate means, in particular by affinity chromatography.

In addition to the foregoing, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the subject of the present invention, with reference to the drawing appended hereto. which: FIG. 1 illustrates the in vitro immunogenicity of 6 HIV-1 peptides;

1 (RT 338-352, Vpr 015-029, Vpr 061-075, Env 031-045, Env 388-402 and Env 602-634) and a mutant of the HIV-I peptide Vif 061-075, exhibiting high affinity for the HLA-DP401 and DP402 molecules, measured by an ELISPOT test, from CD4 + T cell lines from three seronegative individuals: P51, P 123 and P48; T lymphocytes were previously stimulated in vitro by dendritic cells of this same individual loaded with the peptide mixture, then the number of IFN-gamma secretory T cells was measured in the presence of non-L-DP4 cells. charged and loaded with these peptides alone or mixed (Mix) or in the absence of peptides (O). The values indicated correspond to the number of IFN-gamma secretory T lymphocytes per 5000 CD4 + T lymphocytes tested (SFC / 5000 LT).

FIG. 2 illustrates the analysis of the recognition of the native antigen and of the peptides Env 31-45 and RT 338-352 by four lines of CD4 + T lymphocytes specific for said peptides (line 156.66 specific for Env 31-45 and lines 157.49 , 157.53 and 157.13 specific for RT 338-352)), by an IFN-γ assay ELISPOT assay. A: ELISPOT in the presence of autologous immature dendritic cells loaded with the native antigen (Gpl20 or recombinant RT) or the derived peptide (Env 31-45 or RT 338-352; 10 μg / ml) or non-loaded (-). B: ELISPOT in the presence of L-cells DP4 and increasing peptide concentrations (M; 10 ^{^"10-10" 5} M) or without peptide (O). Each value corresponds to the average of two experimental points. ** and * correspond to p <0.01 and p <0.05, respectively.

FIG. 3 illustrates the analysis of the recognition of variants by three lines of CD4 + T lymphocytes specific for Env 31-45 or RT 338 peptides.

352 (156.66 specific line of Env 31-45 and lines 157.53 and 157.13 specific for RT 338-352)), by an ELISPOT assay for IFN-γ assay, in the presence of L-DP4 cells and one of the variants (10 μg / ml) or without peptide (-). Each bar corresponds to the average of two experimental points. ** and * correspond respectively to p <0.01 and p <0.05.

EXAMPLE 1: BINDING ACTIVITY OF HIV PEPTIDES IN RELATION TO HLA-DP401 MOLECULES AND DP402 1) Materials and Methods a) Peptides 13 and 15 amino acid peptides (13-mer and 15-mer) representative of proteins p24, RT, Vif, Vpr and Env of HIV-I were selected according to the presence of several aromatic or hydrophobic residues which are the main anchoring residues for the HLA-DP molecules.

The sequences of the selected peptides are presented in the attached sequence listing and Tables II and III. All peptides correspond to the HXB2 reference sequence (NCBI accession number K03455) excluding the sequences Env 749-763 (SEQ ID NO: 9) and Env 750-762 (SEQ ID No. NO: 31) which correspond to those of the isolate MN (accession number NCBI AF075719), as well as sequences SEQ ID NO: 5 (Vif 61-75) and 27 (Vif 62-74) in which the residues G70 and W71 were reversed with respect to the HXB2 sequence which has the WG and non-GW sequence, at positions 70 and 71 of the Vif protein sequence. This inversion was corrected in the sequences SEQ ID NO: 57 (Vif 61-75) and SEQ ID NO: 58 (Vif 62-74).

In addition, variants of the amino acid peptides RT 338-352 and Env 31-45 correspond to naturally occurring variants of the group M of HIV-I, which are frequently isolated, the sequence of which is available in the LANL database ( http://www.hiv.lanl.gov/content/immunology/map), were also synthesized [SEQ ID NO: 45-52 (Table VIII) and SEQ ID NO: 53-56 (Table IX)].

The peptides were synthesized according to the Fmoc strategy in solid phase parallel synthesis, purified by HPLC and controlled by mass spectrometry (ES-MS). b) HLA-DP4 / peptide binding assay

The binding assays for HLA-DP4 molecules encoded by the DPB1 * 0401 and DPB1 * 0402 alleles, designated DP401 and DP402, respectively, are binding assays in competition with enzyme immunoassay disclosed in PCT International Application WO 03/040299. More specifically, the peptide Oxy 271-287

(EKKYFAATQFEPLAARL, SEQ ID NO: 59) biotinylated at the NH ₂ terminal residue, according to the protocol described in Texier et al., J. Immunol., 2000, 164, 3177-3184), is used as a tracer peptide under the conditions as specified in the Table below. TABLE I: HLA-DP4 binding test conditions

Tracer alleles pH concentration Optimal tracer time of incubation (nM) (h)

DBP 1 * 401 Oxy 271-287 10 5 24 DPB 1 * 402 Oxy 271-287 10 5 24

2) Results

The majority (39 out of 44) of the peptides derived from the HIV-I group M refer ence sequence bind to a good affinity (IC ₅₀ <1000 nM) at one end. at least DP4 molecules (Table II and III). For example, for 15-mer: 17 peptides bind with good affinity to DP401, 15 peptides bind with good affinity to DP402 and 14 peptides bind with good affinity to both DP4 molecules. The 13-mer and the 15-mer bind to DP401 and DP402 with comparable binding activities.

Table II: Binding activities of 15-mer against HLA-DP4 molecules (IC50 in nM)

SEQ

Fr ⁰ "Peptide ID Sequence * DP DP NO: (%) 401 402

822 2739

1 21 RT 263-277 K L N W A S Q I Y P G I K V R ± 106 ± 354

34 31

2 65 RT 338-352 T Y Q I Y Q E F K N L K T G

+ 6 ± 4

424,114

3 8 RT 346-360 F K N L K T G K Y A R M R G A + 35 ± 21

106 45

4 28 RT 403417 T E Y W Q A T W I P E W E F V ± 53 ± 7

5 *** - VIF 061-075 D A R L V I T T Y G W L H T G 142 140

42 52

6 0.1 VPR 061-075 I R I L Q Q L I F I H F F R I

± 16 ± 25

12 7

7 0,4 ENV 388-402 T Q L F N S T W F N S T W S T

± 0 ± 2

1000 2000

8 1 ENV 670-684 W N W F N I T N W L Y Y K L ± 0 ± 0

9 ^** ENV 749-763 VHGFLAIIWVDLRSL 794 1775

2427 3217

10 44 P24029-043 E K A F S P E V I P M F S A ± 850 ± 1550

917 149

11 87 P24 130-144 Y K R W I I L G L N K I V R M ± 1200 ± 28

3744 3947

12 12 RT 005-019 T L N F P I S P I S T V P V K ± 2800 + 212

967,456

13 55 RT 156-170 S P A I E Q S S M T K I L E P ± 177 ± 85

27589 2569

14 58 RT 249-263 K D S W T V N D I Q K L V G K ± 17000 ± 566

630 1849

15 81 RT 408-422 A T W I P E W E F V N T P P L ± 28 + 71

62 57

16 2 VPR 015-029 H N E W T E L L E E L K S E

± 11 ± 11

4 22

17 10 ENV 031-045 T E K L W V T V Y Y G V P W W

+ 0 ± 4

424,194

18 27 ENV 483-497 L Y K Y K V V K I E P L G V A ± 35 ± 71

305,387

19 0.1 ENV 677-691 W L W W Y I K L F I M I V G G + 216 ± 141

173,364

20 1 ENV 684-698 L F I M I V G G L V G L R I V

+ 35 ± 790

624,406

21 21 ENV 760-774 L R S L C L F S Y H R L R D L ± 35 ± 120

35,794

22 0.3 ENV 620-634 E Q I W N H T T W M E W D R E

± 15 ± 141

1332,359

57 ° 7 VIF 061-075 D A R L V I T T Y W G L H T G ± 20 ± 57

* DP4 anchoring residues (Pl, P4, P6 and P9) are indicated in bold ** sequence of isolate MN

*** inversion of G70 and W71 residues compared to HXB2 sequence

° sequence of isolate HXB2

° Frequency of the peptide sequence in the 457 sequences available in the HIV database (Los Alabamos HIV databases http://hiv-web.lanl.gov ^' ).

Table III: Binding activities of 13-mer against HLA-DP4 molecules (IC 50 in nM)

SEQ Conser DP DP ID N "Peptide Sequence * vation 401 402

23 RT 264-276 L N W A S Q I Y P G I K V 4/7 13000 3695

24 RT 339-351 Y Q I Y Q E P K L K T 5/7 92 3,5

25 RT 347-359 K N L K T G K Y A R M R G 2/7 2541 27

26 RT 404-416 E Y W Q A T W I P E W E F 7/7 2655 35

VIF 062-074 A R L V I T T Y G W L H T 3/8 96 5.0

28 VPR 062-074 R I LQ Q L L F I H F F R 6/8 275 6.5

29 ENV 389-401 Q L F N S T W F N S T W S 1/8 64 1,3

30 ENV 671-683 N W F N T N W L W Y I K 2/8 881 537

31 ** ENV 750-762 H G F L A I I W V D L R S 3/6 5224 24

32 P24 030-042 K A F S P E V I P M F S A 8/9 15000 1407

33 P24 131-143 K R W I I L G L N K I V R 8/9 6618 28

34 RT 006-018 L N F P I S P I S T V P V 6/7 5514 647

35 RT 157-169 P A I E Q S S M T K I L E 6/7 2814 28

36 RT 250-262 D S W T V N I K L V G in 8500 212

37 RT 409-421 T W I P E W E F V N T P P 6/7 5000 453

38 VPR 016-028 N E W T E L L E L K S 5/8 28 OJ

39 ENV 032-044 E K L W V T Y Y G V P W 7/8 8.7 3.5

40 ENV 484-496 Y K Y K V V K I E P L G V 4/8 1568 68

41 ENV 678-690 L W W Y I K L F I M I V G 6/8 272 105

42 ENV 685-697 F I M I V G G L V G L R I 7/8> 10000 57

43 ENV 761-773 R s L C L F S Y H R L R D 5/8> 10000 351

44 ENV 621-633 Q I W N H T T W M E W D R 1/8 30 150

* DP4 anchoring residues (Pl, P4, P6 and P9) are shown in bold ** sequence of isolate MN

*** inversion of G70 and W71 residues compared to HXB2 sequence

Immunogenicity tests were performed with six or seven high affinity peptides for DP401 and DP402 (RT 338-352, Vpr 015-029, Vpr 061-075, Env 031-045, Env 388-402 and Env 620 peptides). -634, with or without the mutant of Vif 061-075 (SEQ ID NO: 5)).

EXAMPLE 2 IMMUNOGENICITY OF IN VITRO HIV PEPTIDES

The ability of peptides with good affinity for molecules

HLA-DP4 to induce in vitro stimulation of specific T lymphocytes was evaluated from blood samples of HIV-negative people. The aim is to evaluate the ability to recruit CD4 + precursor are in a naïve individual at a very low frequency, that is to say to perform an in vitro immunization using these peptides. 1) Materials and methods a) Individuals tested

Blood samples were taken from eight HLA-DP4 positive and HIV-negative individuals. The alleles of the DPB1 gene of these individuals are shown in Table IV.

Table IV: DPBl Alleles of Individuals Tested

b) Obtaining HIV-specific CD4 + T lymphocyte lines restricted to HLA-DP4

Mononuclear peripheral blood cells (PBMC) were separated on a Ficoll gradient. The PBMCs were then cultured in AIM V medium (LIFE TECHNOLOGIES) and incubated in flasks in an oven at 37 ° C. in the presence of 5% CO ₂ . After one night of incubation, the non-adherent cells were recovered, and then the CD4 + T cells were purified with magnetic beads coated with anti-CD4 antibodies (LS + column, MYLTENYI) and frozen. The adherent cells were incubated for 5 days in AIM-V medium containing 100 μ / ml of GM-CSF and 100 μ / ml of recombinant IL-4 (R & D SYSTEMS), then the cells differentiated into dendritic cells (immature dendritic cells). ) were then cultured for 2 days, in the presence of 1 μg / ml of LPS, 1000 U / ml of IL-4 and 1000 U / ml of GM-CSF, so as to induce their maturation. The mature dendritic cells (10,000 cells / well) were then incubated with the peptide mixture (10 μg of each peptide in IMDM medium (IN VITROGEN) supplemented with glutamine (24 mM, SIGMA), asparagine (55 mM, SIGMA), arginine (150 mM, SIGMA), penicillin (50 IU / ml, IN VITROGEN), streptomycin (50 μg / ml, IN VITROGEN) and 10% human serum) for 4 hours at 37 ^° C. The mature dendritic cells were then washed and then incubated, in the presence of the previously thawed CD4 + T lymphocytes (100,000 cells / well), in medium containing 1000 U / ml of IL-6 and 10 ng / ml of IL-12. After 7 days (J7), the culture was restimulated a first time, using mature dendritic cells previously thawed and loaded with the peptide mixture, in medium containing IL-2 (10 U / ml) and IL-7 (5 ng / ml). After two additional stimulations (J14 and J21), under the above conditions, the cells were tested in ELISPOT, at least 6 days after the last stimulation. c) Analysis of the specificity of ELISPOT lines

Anti-IFN-gamma antibodies (1-D1K, MABTECH) diluted to 1 μg / ml in PBS buffer were adsorbed onto nitrocellulose (MILLIPORE) plates for 1 hour at 37 ° C. The plates were then washed with PBS and then saturated with ISCOVE medium containing 10% human serum of the AB group (100 μl / well), for 2 h at 37 ^° C. The antigen presenting cells are either cells immature autologous dendritics prepared as specified above, namely HLA-DP4 positive cells (L-DP4 line of fibroblasts transfected with the cDNA coding for a DP402 molecule; Yu et al., Hum Immunol., 1990, 27, 132- 135) which makes it possible to check the specificity of the peptides with respect to the HLA-DP4 molecule. The recombinant HIV proteins (RT of HIV-I isolate HXB2 or HIV-IIB GP 120, NIBSC, United Kingdom) are incubated with immature dendritic cells for 4 h at 37 ^° C. and the dendritic cells are washed. before being used. Peptides are added directly to the plates. The dendritic cells (2x10 ⁴ cells / well) or the L-DP4 cells (3 × 10 ⁴ cells / well) are added to the plates with CD4 + T lymphocytes (2x10 ³ to 10 ⁴ cells / well) and incubated overnight at 37 ° C. C, in the presence or absence of a single peptide, a mixture of peptides or recombinant HIV protein. After a washing with 0.05% PBS Tween buffer and then with PBS alone, 100 μl of biotin-conjugated anti-IFN-γ secondary antibody (7-B6-1-biotin, MABTECH), diluted to 0.25 μg / ml in PBS containing 1% BSA, was added to each well. After one hour of incubation, the plates were washed again and incubated with 100 .mu.l / well of Extravidin-AKP (E-2636, SIGMA) diluted 1/6000. After washing the plates in PBS buffer, 100 μl of NBT / BCIP substrate (B-5655, SIGMA) diluted in water (1 tablet in 10 ml of water) was dispensed into each well. Immunoenzymatic revelation was stopped after about 10 minutes, by extensive rinsing of the plates in water, and the colored spots were counted using a PLC (AID). Restriction control comprises T cell lines and antigen (peptide (s) or HIV protein) without L-DP4 cells. The experimental points were duplicated. The statistical analysis was performed using the t test. A response is considered positive when it is at least three times higher than that obtained with control without peptide. 2) Results a) Induction of HIV specific CD4 + T lymphocyte lines by DP4 ligand peptides

25 and 23 HIV-specific and HLA-DP4 restricted lines were demonstrated when the mixture of seven and six peptides, respectively, for stimulation was used; these lines are stimulated by the mixture of peptides (mix) presented by HLA-DP4 but not in the absence of the mixture or HLA-DP4 (Tables V and VI, Figure 1).

Table V: Immunogenicity of Peptides *

* Stimulation with the mixture of 7 peptides: RT 338-352, Vpr 015-029, Vpr 061-075, Env 031-045, Env 388-402, Env 620-634 and Vif mutant 061-075 (SEQ ID NO: 5) ** five donors were tested: P48, P51, Pl 19, P120 and P123 Table VI: Immunogenicity of the peptides

* Stimulation with the mixture of 6 peptides: RT 338-352, Vpr 015-029, Vpr 061-075,

Approx 031-045, Env 388-402 and Env 620-634

** eight donors were tested: P48, P51, Pl19, P120, P123, P147, P156 and P157

Each line responds to at least one of the peptides of each of the mixtures. Five peptides are recognized by at least one of the lines (RT 338-352, Env 031-045, Env 388-402, Env 620-634 and mutant Vif 061-075 (SEQ ID NO: 5)), indicating that at least five of the seven peptides tested are immunogenic in an HLA-DP4 context (Tables V and VI).

Of these peptides, two are highly conserved between the different subtypes of the M group of HIV-I (RT 338-352 and Env 31-45), one peptide is conservatively stored (Vif 061-075) and the other two (Env 388-402 and Env 620-634) are poorly preserved (Table VII).

Table VII: Preservation of peptides within group M

b) recognition of native antigen by CD4 + T cell lines specific for HIV peptides

The recognition of the native antigen by the CD4 + T lymphocyte lines specific for the Env 31-45 or RT 338-352 peptides was analyzed by an ELISPOT assay for PIFN-γ assay, in the presence of immature autologous dendritic cells loaded with 1 native antigen (Gp 120 or recombinant RT) or the derivative peptide (Env 31-45 or RT 338-352), or uncharged. Three CD4 + T lymphocyte lines are specific for the RT 338-352 peptide (lines 157.49, 157.53 and 157.13, FIG. 2A); these lines are stimulated by dendritic cells loaded with reverse transcriptase (RT) but are not stimulated by cells dendritic loaded with envelope protein (Env or Gp120) or derivative peptide (Env 31-45). In contrast, a CD4 + T cell line is specific for the Env 31-45 peptide (line 156.66, Figure 2A); this line is stimulated by dendritic cells loaded with envelope protein (Env or Gp120) but is not stimulated by dendritic cells loaded with reverse transcriptase (RT) or derivative peptide (RT 338-352). The concentration of peptide making it possible to obtain a stimulation of the lines equal to 50% of the maximum stimulation, is of the order of 10 ^-8 to 10 ^-7 M (FIG. 2B).

The HLA-DP4 ligand peptides are immunogenic as they induce HIV-specific CD4 + T cell lines capable of

H to be effectively stimulated in their presence and to recognize the epitope of the native antigen naturally presented by the dendritic cells. c) Recognition of HIV variants by peptide-specific CD4 + T cell lines

The effect of natural HIV mutations on peptide binding to HLA-DP4 and stimulation of CD4 + T cells was analyzed for RT 338-352 and Env 31-45 (Tables VIII and IX, Figure 3).

Table VIII: Binding activities of the variants * of the RT 338-352 peptide with respect to the HLA-DP4 molecules (IC ₅₀ in nM)

SEQ ID

DP DP NO: Variant of

Sequence ** RT 338-352 401 402

55 35

45 G352R T Y Q I Y Q E F K N L K T R ± 23 ± 0

30 45

46 K350R T Y Q I Y Q E F K N L R T G

± 1 ± 7

35 23

47 N348I T Y Q I Y Q E F K I L K T G

+ 5 ± 23

160,145

48 F346Y T Y Q I Y Q E P Y K N L K T G ± 64 ± 7

49 F346H T Y Q I Y Q E P K L K T G> 10000> 10000

88 44

50 P345T T Y Q I Y Q E K L K T G ± 36 ± 27

50 58

51 P345Q T Y Q I Y Q E Q K L K T G ± 32 ± 5

90 56

52 Y342F T Y Q I F Q E F K N L K T G ± 4 ± 34 * mutated residues are underlined

** DP4 anchoring residues (Pl, P4, P6 and P9) are shown in bold Table IX: Binding Activities of Env 31-45 Variant * Versus HLA-DP4 Molecules (IC ₅₀ in nM)

SEQ

Variant of DP DP ID NO: Sequence ** Env 31-45 401 402

53 T31A, K33N, W44V A E N L W V T Y Y G V P V W 9 32

T31A, K33Q,

54 W44V A E Q L W V T V Y Y G V P V W 4 10

T31V, E32G,

55 K33N, W44V V G N L W V T V Y Y G V P V W 10 36

T31 M, E32G,

56 K33N, W44V M G N L W V T V Y Y G V P V W 9 34

* mutated residues are underlined

** DP4 anchoring residues (Pl, P4, P6 and P9) are shown in bold. The RT 338-352 peptide is highly conserved; of the 457 sequences analyzed, 298 sequences (65%) are identical to those of isolate HXB2. The analysis of the HLA-DP4 binding activity of 8 of the most frequent variants among the 47 variants listed (Table VIII), shows that all the variants are likely to be presented by an HLA-DP4 molecule at the same time. exclusion of variant F346H (or 346H) which has a histidine residue unfavorable to anchorage in the P6 pocket. Two variants (K350R and G352R or 350R and 352R) are capable of stimulating both CD4 + T cell lines specific for the HXB2 sequence, and four variants (N348I, F346H, F346Y and Y342F or 3481, 346H, 346Y and 342F). are able to stimulate one of them (Figure 3). Overall analysis of the Env 31-45 peptide sequence indicates that it is not conserved since the HXB2 sequence is present in only 3.9% of the isolates analyzed. However, a more detailed analysis shows that the central region of this peptide (positions 34-42) which comprises the sequence located between the anchoring residues P1 and P9 is highly conserved and present in 80% of the isolates analyzed. The activity of the binding of the variants of the sequences flanking the central region shows that all the tested variants (NEA, AEQ, VGN, MGN, SEQ ID NO: 53 to 56) are likely to be presented by an HLA-DP4 molecule ( Table IX) and to stimulate specific T cells of the HXB2 sequence (Figure 3).

These results indicate that the RT338-352 and Env 31-45 peptides are capable of inducing CD4 + T cells capable of recognizing HIV variants.

Claims

1) HIV peptide comprising a CD4 + T epitope capable of being expressed by an HLA-DP4 molecule and of inducing HIV-specific CD4 + T lymphocytes, selected from the group consisting of: a) fragments of 13 consecutive amino acids Env proteins,

RT, Vpr and Vif located between the following positions:

16 to 28 and 62 to 74 of the Vpr protein, and

- 62 to 74 of the protein Vif, b) the fragments of 14 or 15 consecutive amino acids of said proteins RT, Env, Vif and Vpr, which include the positions defined in a), and c) the variants of the peptides defined in a) or b), which have an identical length and a binding activity to an HLA-DP4 molecule less than or equal to 1000 nM, for use as an antigen in prophylactic or therapeutic vaccination, or the diagnosis of infection with HIV.

2) Peptide according to claim 1, characterized in that its sequence corresponds to that of a type 1 HIV isolate.

3) A peptide according to claim 2, characterized in that said sequence corresponds to the reference sequence HXB2, having the access number NCBI K03455.

4) The peptide according to any one of claims 1 to 3, characterized in that it is selected from the group consisting of the peptides RT 338-352, Env 31-45, Env 388-402 and Env 620- 634.

5) Peptide according to any one of claims 1 to 4, caracté¬ ized in that it is selected from the group consisting of the sequences SEQ ID NO: 2 to 7, 16 to 20, 22, 24 to 29, 38 to 42, 44 to 48 and 50 to 56. 6. Peptide according to any one of claims 1 to 5, characterized in that it is marked or complexed. 7. Peptide according to claim 6, characterized in that it is complexed with labeled HLA-DP4 molecules.

8) A polyepitopic fragment comprising the concatenation of at least two identical or different epitopes, at least one of which is a peptide as defined in any one of claims 1 to 5, for use as an antigen in prophylactic vaccination or therapy, or the diagnosis of HIV infection.

9) polyepitopic fragment according to claim 8, characterized in that it comprises the concatenation of at least one HIV CD4 + T epitope as defined in any one of claims 1 to 5 and an epitope selected from the group consisting of by :

- T CD 8+ epitope of an HIV protein ₅

a CD4 + T epitope of the HIV Nef protein, in particular Nef 1-36, 66-94, 74-88, 77-91, 137-168, 139-153, 175-190, 182-198, 178-192, 181-195, 183-197, 187-201 or 189-203,

a natural or synthetic universal CD4 + T epitope such as the peptide TT 830-846, PADRE, the peptide 45-69 of the Nef protein of HIV-I and the peptides CS.T3, CSP, SSP2, LSA-I and EXP- I

an epitope B consisting of a sugar, preferably in the form of a glycopeptide, and / or

an epitope B of an HIV protein.

10 °) fusion protein comprising a protein or a fragment of protein, fused to a peptide as defined in any one of claims 1 to 5 or a polyepitopic fragment as defined in claim 8 or claim 9, for use as an antigen in prophylactic or therapeutic vaccination or diagnosis of HIV infection.

11 °) fusion protein according to claim 10, characterized in that said protein or said fragment is selected from the group consisting of an alpha or beta chain of an HLA-DP4 molecule or a fragment of said chain corresponding to an HLA molecule -DP4 soluble.

12 °) lipopeptide obtained by addition of a lipid on an α-amino function or a reactive function of the side chain of a peptide as defined in any one of claims 1 to 5 or a polyepitopic fragment according to claim 8 or claim 9 for use as an antigen in prophylactic or therapeutic vaccination or diagnosis of HIV infection. 13 °) Expression vector comprising a polynucleotide encoding a peptide as defined in any one of claims 1 to 5, a polyepitopic fragment as defined in claim 8 or claim 9 or a fusion protein as defined in claim 10, under the control of appropriate regulatory sequences, transcription and, optionally, translation, for use as an antigen in prophylactic or therapeutic vaccination against HIV infection.

14 °) Immunogenic or vaccinal composition, characterized in that it comprises at least one peptide as defined in any one of revendi¬ cations 1 to 5, a polyepitopic fragment as defined in claim 8 or claim 9, a lipopeptide as defined in claim 12 or an expression vector as defined in claim 13, and a pharmaceutically acceptable carrier, and / or a carrier substance, and / or an adjuvant.

15. Immunogenic composition according to claim 14, caractéri¬ sée in that it comprises at least one peptide as defined in any one of claims 1 to 5 and another peptide including an HIV CD8 + T epitope, or a polyepitopic fragment comprising said peptides, as defined in revendica¬ tion 9 or an expression vector encoding said peptides or said fragment, as defined in claim 13.

16. Immunogenic composition according to claim 15, characterized in that it comprises at least one peptide including an epitope selected from the group consisting of a CD4 + T epitope of the HIV Nef protein, a universal CD4 + T epitope, a epitope B constituted by a sugar and an epitope B of HIV, as defined in claim 9.

17 °) Use of a peptide as defined in any one of Claims 1 to 5, of a polyepitopic fragment as defined in Claim 8 or Claim 9, of a lipopeptide as defined in Claim 12 or an expression vector as defined in claim 13, for the preparation of a vaccine for the prevention and / or treatment of HIV infection in an HLA-DP4 positive individual.

18 °) Use of a peptide as defined in any one of claims 1 to 5, for the preparation of a diagnostic reagent for the immune status of an individual HLA-DP4 positive, vis-à-vis HIV.

19 °) Method for diagnosing the immune status of an individual with respect to HIV, characterized in that it comprises:

contacting a biological sample of said individual with a peptide as defined in any one of Claims 1 to 7, and detecting antigen-specific CD4 + T lymphocytes.

HIV.

20 °) A method for sorting CD4 + T lymphocytes specific for an HIV antigen, characterized in that it comprises at least the following steps:

contacting a cell sample with marked multimeric complexes formed by the binding of soluble HLA-DP4 molecules with at least one peptide as defined in any one of Claims 1 to 7, and

sorting the cells bound to said HLA-DP4 / peptide complexes.

21 °) A peptide as defined in any one of claims 1 to 7, excluding peptides RT 338-352, RT 403-417, Env 483-497, Env 684-698 and Vpr 15-29.

22 °) polyepitopic fragment comprising the concatenation of at least two identical or different epitopes of which at least one is a peptide according to claim 21.

23 °) fusion protein comprising a protein or a fragment of protein, fused to a peptide according to claim 21 or a poly¬ epitopic fragment according to claim 22.

24 °) A polynucleotide encoding a peptide according to claim 21, a polyepitopic fragment according to claim 22 or a fusion protein according to claim 23. 25 °) Expression vector comprising a polynucleotide according to claim 24 under the control of regulatory sequences appropriate, transcription and possibly translation. 26 °) host cell modified with a polynucleotide according to revendi¬ cation 24 or an expression vector according to claim 25.