WO1999066046A1

WO1999066046A1 - Hiv virus mimotopes

Info

Publication number: WO1999066046A1
Application number: PCT/FR1999/001409
Authority: WO
Inventors: Véronique BARBAN
Original assignee: Aventis Pasteur
Priority date: 1998-06-12
Filing date: 1999-06-14
Publication date: 1999-12-23
Also published as: AU4148999A; NZ508812A; CA2331197A1; EP1084253A1; AU748700B2

Abstract

The invention concerns a peptide for therapeutic or prophylactic treatment of HIV infection, capable of reacting with an antibody specific of the HIV virus envelope and derived from a HIV positive patient belonging to the long term non-progressor group, comprising an amino acid sequence mimicking an antigen conformational epitope of said virus envelope without however corresponding to a continuous sequence of said antigen and capable of containing optionally one of the 11 amino acid sequences as specified in the description. The invention also concerns a peptide conjugate obtained by combining the peptide with a carrier molecule for reinforcing said peptide immunogenicity. The invention further concerns a recombinant vector comprising a functional expression cassette for expressing a polynucleotide coding for said peptide. The invention likewise concerns a therapeutic or prophylactic composition for HIV infection, in particular for vaccinal use, whereof the principle comprises said peptide, or as the case may be said peptide conjugate and/or a recombinant vector coding for said peptide. Finally the invention concerns the use of said peptide as reagent for diagnosing HIV infection and/or the proneness of subjects in contact with the virus for rapidly developing AIDS.

Description

HIV virus mimotopes

Subject of the invention

The present invention relates to the treatment and prevention of the HIV virus and in particular to any peptide mimicking new conformational epitopes of antigens of the envelope of the HIV virus and to any polynucleotide integrated in a vector allowing the expression of said peptides and their use for therapeutic, prophylactic, in particular vaccine, and / or diagnostic purposes.

Field of the invention

HIV is an RNA-enveloped virus and represents the etiological agent of acquired immunodeficiency syndrome or AIDS, the outcome of which is fatal in the long term, characterized by progressive destruction of the immune system and the concomitant development of micro-biological infections causing often due to opportunistic germs.

The majority of individuals develop acquired immunodeficiency syndrome within 10 years of being infected. Indeed, the immune reactions in response to infection are very often inadequate and in particular those which are directed against the envelope proteins because of the very great variability of the virus resulting from its great capacity to multiply, mutate and to recombine (Bangham CRM et al 1997 Lancet 350: 1617-1621). The great variability of the HIV virus makes it possible to escape control by the immune system and thus promotes the spread of the virus. However, recent epidemiological studies have shown that some infected people can contain their infection, without apparent clinical manifestations and without biological signs of immunosuppression for periods of time greater than 10 years (Pilgrim AK et al., 1997, J. Infect Dis 176: 924-932). The serum of these people also called "long-term non progressors" reveals the presence of neutralizing antibodies against primary isolates of HIV virus (Piigrim A.K et al., 1997, J. Infect. Dis. 176: 924-932).

The envelope of the HIV virus, derived from the expression product of the Env gene (envelope gene), is first synthesized in the form of a glycoprotein Gp160 which then cleaves into two glycoproteins Gp 120 and Gp 41 These 3 proteins are found on the surface of cells infected with HIV. In addition, Gp 160 and Gp 120 have an affinity for the CD4 molecule present on the surface of certain T lymphocytes, a molecule CD4 which acts as a gateway for the HIV virus to enter the cell. So far, very few epitopes accessible to the immune system have been described on the envelope of the HIV virus (Burton DR, 1997 Proc.Natl. Acad. Sci. USA 94: 10018-10023).

There is therefore a need in the characterization of new epitopes of the HIV envelope.

There is also a need to identify envelope epitopes which are inducers of neutralizing antibodies, or in other words which are capable of reducing or suppressing viral spread.

There is also a need to identify a pharmaceutical composition which can effectively treat or prevent HIV virus infection.

Finally, there is also a need to develop reagents which are particularly used in the composition of immunological kits which make it possible in particular to distinguish among infected persons those which are most resistant to infection or "long-term non progressor" and to test, by example the effectiveness of new vaccines by their ability to induce neutralizing antibodies.

Summary of the invention

The present invention aims to overcome these needs by identifying new peptide structures for the therapeutic or prophylactic treatment of HIV infection from the use of a combinatorial library of antibodies from HIV positive patients and belonging to the group. "long term non progressor". To this end, the invention relates to any peptide structure capable of reacting with an antibody specific for an antigen of the HIV envelope originating, for example, from a combinatorial library of antibodies obtained from lymphocytes of HIV positive patients. and belonging to the group of "long term non progressors", comprising an amino acid sequence which mimics a conformational epitope of the envelope of said virus without however corresponding to a continuous amino acid sequence of this antigen.

The selection of mimotopes using recombinant antibodies from HIV positive patients and belonging to the group of "long term non progressor" presents the advantage of identifying new neutralizing antibody-inducing epitopes which are effective in protecting against primary isolates of HIV virus

The present invention also relates to any recombinant vector comprising a functional expression cassette allowing the expression of a polynucleotide coding for a peptide meeting the criteria defined above.

The present invention also relates to a therapeutic or prophylactic composition of the HIV virus, in particular intended for vaccine use, the active principle of which comprises a peptide meeting the criteria defined above and / or a recombinant vector coding for said peptide.

Finally, the present invention also relates to

the use of a peptide meeting the criteria defined above as a reagent for the diagnosis of the HIV virus making it possible in particular to identify subjects in contact with the virus more resistant to infection, said diagnosis comprising the evaluation, from a blood sample, the humoral and / or cell-mediated response specific for this peptide.

the use of a peptide meeting the criteria defined above and / or of a recombinant vector coding for said peptide for the preparation of a therapeutic or prophylactic composition intended for the treatment or the prevention of infection by the virus HIV.

Description of the invention

In the context of the present invention, various terms used are defined below:

"Peptide" means a sequence of at least 6 amino acids linked together by a peptide bond obtained by chemical synthesis or by genetic recombination techniques, preferably between 6 and 100 amino acids and in particular between 20 and 80 amino acids .

"By HIV envelope antigen is meant any molecular entity that binds to an antibody specific for any product derived from the env gene, comprising in particular gp 160 in natural or recombinant form, its derivatives constituted by gp41 and gp 120 may also be in natural or recombinant form and the products derived from the combination of said molecules.

"By conformational epitope" means a three-dimensional structure which allows its positioning in the specific binding site of an antibody, like a key in a lock, and which is represented by an amino acid sequence which does not correspond not a continuous amino acid sequence of the protein against which this antibody is raised. Preferably, this amino acid sequence of the conformational epitope is not homologous to a continuous amino acid sequence of the natural or recombinant protein, the homology being defined by the combination of two criteria:

the amino acid identity criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are identical to those of a sequence of the same size carried by the natural or recombinant protein, and the total number of amino acids of said peptide. Preferably the identity in amino acids will preferably not exceed 50%, even 60% or 70% or 80% or even 90%. - The linking criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are both identical and are in the same linking position as those of a sequence of the same size carried by the natural or recombinant protein, and the total number of amino acids of said peptide. Preferably the linking identity will not exceed 70 to 80%.

"Long term non progressor" means HIV positive subjects characterized on the clinical level, in that they do not develop AIDS since they are contaminated with a decline of at least ten years, on the biological level, in what they do not show signs of immunosuppression with in particular a rate of CD4 T lymphocytes higher than 600 / mm ³ and finally not receiving any particular antiviral treatment.

"By mimotope" means an epitope which mimics the three-dimensional structure of another epitope by binding to the specific binding site of the same antibody

"By CDR3" means the hypervariable amino acid chain region of the heavy and light chain of immunoglobulins and which is located at the site of specific interaction with the epitope.

"Conjugate" means the association of the peptide as defined in the invention with any other molecule, by physical or chemical methods, intended to induce or strengthen the immunogenicity of the starting peptide.

"Immunogenicity" means the ability of a molecular entity, after inoculation into a mammal, to induce production of antibodies specifically directed against this entity.

"Polynucleotide" means either an RNA sequence, or a DNA sequence, or a cDNA sequence resulting from the reverse transcription of a sequence of natural or synthetic origin, with or without modified bases.

"Mucosal route" means a mode of administration which brings the pharmaceutical composition directly into contact with the different types of mucous membranes in the body.

"Parenteral route" means a mode of administration which puts the pharmaceutical composition directly in contact with the internal tissues or organs of the organism.

The invention therefore relates to any peptide which mimics a conformational epitope of an antigen of the HIV envelope and which is recognized by an antibody obtained from a "long term non progressor" patient and specific for this antigen. A peptide according to the invention can be advantageously represented by one of the 11 sequences as follows

SEQ ID NO: 1 Phe Asn Leu Thr His Phe Leu SEQ ID NO: 2 Glu Gly Trp His Ala His Thr

SEQ ID NO: 3 Lys Leu Asn Trp Met Phe Thr

SEQ ID NO: 4 Ser Thr Asn Trp Met Phe Thr

SEQ ID NO: 5 Ala Met Pro Leu Pro Tyr Thr Phe

SEQ ID NO: 6 Asp Ser His Thr Pro Gin Arg SEQ ID NO: 7 Val Ser Phe Thr Pro Ser Phe SEQ ID NO: 8 His Ala Ala Leu Ser Met Asn Thr His Ala Leu Met

SEQ ID NO: 9 Ala Trp His Glu Ser Arg Ala

SEQ ID NO: 10 Phe Lys Thr Ala Tyr Pro Thr

SEQ ID NO: 11 Ser His Ala Leu Pro Leu Thr Trp Ser Thr Ala Ala

From a combinatorial bank of antibodies obtained in particular from the peripheral blood of a subject having been infected with the HIV virus and belonging to the group of "long-term non progressors", characterized in that said subject belonging to this group has been asymptomatic on the clinical level for at least 10 years and does not show biological signs of immunosuppression with in particular a CD4 T lymphocyte level greater than 600 / mm ³ , and a synthetic library of peptides, it can be identified, by means of mimotopes, new conformational epitopes present on the envelope of HIV, in particular epitopes which can be located outside the V3 loop, such as for example epitopes located in the region of the site of binding to the CD4 receptor, epitopes spanning the V2 loop and the CD4 receptor binding site or even epitopes spanning the C2, C3 and V4 regions of Gp120 or epitopes located in non-immune regions odomants from Gp 41 (clusters I and II).

The identification of these mimotopes requires for their implementation a sophisticated technological process, namely:

-the constitution of a combinatorial library of antibodies sufficiently complex to best reflect the natural antibody repertoire of an individual, and advantageously the antibody repertoire of an individual infected with HIV and belonging to the group of "long -term non progressors ";

the selection from this bank of recombinant antibodies specific for antigens of the HIV envelope, in particular expressed by Gp160, Gp120, Gp41 can be in the form of natural or recombinant proteins, glycosylated or deglycosylated, monomeric or multimeric or finally combined or not. The selection of specific recombinant antibodies preferably comprises an additional step consisting in measuring the neutralizing activity of these recombinant antibodies with respect to viral infection mediated in particular by one or more primary isolates of the HIV virus. Preferably, specific recombinant antibodies which neutralize several primary isolates of the HIV virus.

the selection of specific peptides for the recombinant antibodies, from a random synthetic library of peptides obtained by molecular recombination, the said selection being able to be made by ELISA;

the characterization of these peptides as being mimotopes of conformational HIV epitopes in that there is no correspondence between the amino acid sequence of said peptide with any continuous amino acid sequence found in the proteins of l HIV envelope on the one hand and on the other hand in that this peptide is capable of inhibiting the interaction of the recombinant antibody with the product of the env gene which served for the selection of said recombinant antibody.

It is also possible to transform the lymphocytes originating from a "long term non progressor" patient by using, for example the Epstein virus

Barr (EBV) for the selection of new monoclonal antibodies specific for the HIV envelope. The lymphocyte transformation process is well known to those skilled in the art and results, after several cycles of selection against the antigen of interest, in obtaining the transformed and immortal lymphocyte clones, each clone producing a single type of monoclonal antibody . These monoclonal antibodies, like recombinant antibodies from the combinatorial library, can also be tested for their neutralizing activity against various primary HIV isolates before being used for the selection of mimotope peptides from the HIV envelope.

To induce or rather reinforce the immunogenicity of the peptide mimicking a conformational epitope of HIV, the present invention also relates to peptides comprising a repetition (2 or more) of the peptide according to the invention. In particular, the coupling of the two identical peptides can be done if necessary by means of an intermediate spacer arm constituted by the chain of amino acids Gly Pro Gly.

The invention also relates to a combination of different peptides according to the invention, as well as to peptides comprising both repeats and combinations. In such cases, the peptides can be joined by covalent bonds or non-covalent bonds. For example, one can advantageously cite the method developed by Posnett et al (J. Biol. Chem. (1988) 263: 1719) which does not alter the three-dimensional structure of the epitope or epitopes carried by the peptide and results in the formation of multimers of the same peptide or of different peptides.

In the context in particular of the antigenic preparations and vaccine formulations which are described below, it is also possible to prefer to conjugate, by covalent bonding, the peptides of the invention to immunogenic molecules usually used to make small peptides immunogenic.

The peptides according to the invention can thus be conjugated to known immunogenic proteins such as serum albumin, thyroglobulin, ovalbulmine, gelatin, haemocyanin (eg Keyhole Limpet Haemocyanin KLH), seroglobulins, tetanus toxoid, diphtheria toxoid, proteins from external membranes of bacteria, etc, but it is also possible to prefer to conjugate the peptides with “T helper” epitopes among which the “T helper” epitopes of HIV are chosen, for example the T1 and p24E epitopes as described in WO 94/29339 (Connaught). The conjugation reactions with said "T helper" epitopes of HIV allow us to obtain compounds whose sequential sequence is p24E-GPG-X-GPG-T1 where:

-p24E symbolizes the amino acid sequence of the p24E epitope, said sequence being placed on the N-terminal side of the peptide according to the invention; -GPG symbolizes the glycine-proline-glycine sequence;

-X symbolizes the peptide of interest according to the invention which may be, if necessary, the product of the combination of several identical or different amino acid sequences in accordance with the invention;

-T1 symbolizes the amino acid sequence of the T1 epitope, said sequence being placed on the C-side of the peptide according to the invention;

Among these compounds, mention may be made of the compounds comprising the sequences 12 to 14

SEQ ID NO: 12 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Gly Pro Gly Lys Leu Asn Trp Met Phe Thr Gly Pro Gly Lys Leu Asn Trp Met Phe Thr Gly Pro Gly lys Gin Ile Ile Asn Met Trp Gin Glu Val Glu Lys Ala Met Tyr Ala

SEQ ID NO: 13 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg

Phe Tyr Lys Gly Pro Gly Ser Thr Asn Trp Met Phe Thr Gly Pro Gly Ser Thr Asn Trp Met Phe Thr Gly Pro Gly lys Gin Ile Asn Met Trp Gin Glu Val Glu Lys Ala Met Tyr Ala

SEQ ID NO: 14 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg

Phe Tyr Lys Gly Pro Gly Phe Asn Leu Thr His Phe Leu Gly Pro Gly Phe Asn Leu Thr His Phe Leu Gly Pro Gly Lys Gin Ile Asn Met Trp Gin Glu Val Glu Lys Ala Met Tyr Ala

These conjugates can themselves be grafted onto a branched lysine framework so as to obtain polymers of said conjugates in a branched form as described in WO 94/29339 (Connaught), the technical content of said patent being incorporated by reference into the subject matter of the invention.

Conjugation techniques are also perfectly known to those skilled in the art. Heterobifunctional agents such as SPDP, carbodiimide, glutaraldehyde, biotin / avidin system, etc. can be used, for example.

The peptides can also be coupled to lipopolysaccharides, polysaccharides, glycopeptides, muramyl peptide analogs, fatty acids, etc. Preferably, a peptide is coupled with a fatty acid of the palmitoyl-lysine type as described in EP 491628 (Biovector) or (Pam) 3

Cys-Ser as described in EP547681 (Merck), for example, the technical content of said patents being incorporated by reference into the subject of the invention.

Methods for operably linking peptides individual by side chains carrying amino acid residues, in order to form an immunogenic conjugate, for example a branched polypeptide polymer, are also well known to those skilled in the art. By these methods, it is sought to establish links on different side chains by one or more types of functional groups in order to obtain a structure in which the peptide structures are covalently linked while being separated by at least one side chain. As functional groups, there may be mentioned the epsilon amino groups, the beta- or gamma-carboxylic groups, the thiol groups (-SH) and the aromatic rings (for example tyrosine and histidine). Methods for binding polypeptides using these functional groups are described in Erlanger (1980 Method of Enzymology, 70: 85), Aurameas et al., (1978 Scand. J. Immunol., Vol.8, suppl. 7 , 7-23) and US-A-4,193,795. In addition, it is also possible to carry out a directed coupling reaction as described in Rodwell et al., (1985 Biotech 3, 889-894). The peptides can also be modified to incorporate spacer arms such as hexamethylene diamine or other bi-functional molecules of similar sizes.

The peptides can also be formulated with alum, monophosphoryl Lipid A, pluronics, SAF1, Ribi, treha! Ose-6,6-dimycolate or other immunostimulatory compounds known to those skilled in the art to increase immunogenicity of the peptide to which these compounds are linked.

However, all these methods of conjugation, modification, repetition or combination of peptides in accordance with the invention must best respect the original conformation of the peptide.

The present invention also relates to the DNA fragments coding for the peptides according to the invention and which can be used to produce the peptides by expression of the DNA sequence in an appropriate expression system. Taking into account the degeneracy of the code, those skilled in the art are perfectly capable of determining the different DNA sequences capable of coding for the different peptides in accordance with the invention. According to a first aspect of the invention, the expression system is an in vitro expression system for the production of the peptides with a view to their subsequent use, eg as a diagnostic reagent, as an antigenic component or as a vaccine component. Such systems or vectors for in vitro expression are perfectly known to those skilled in the art and mention may be made, for example, of bacteria such as E. coli, eukaryotic cells such as yeasts, in particular S. cerevisiae. , baculovirus, in particular spread on insect cells, etc.

The invention therefore also relates to an expression cassette comprising such a DNA fragment and regulatory sequences allowing the expression of this DNA fragment in an appropriate in vitro expression system.

According to a second aspect of the invention, the expression system is an in vivo expression system for generating an immune reaction, preferably protective, in the patient treated. In other words, the expression system, which may be replicative or non-replicative, will express the peptide in vivo. Those skilled in the art have at their disposal such systems. By way of preferred examples, mention may be made of plasmids, in particular naked plasmids, eg according to WO-A-90 11092, WO-A-93 19813, WO-A-94 21797 and WO-A-95 20660, poxviruses, such as vaccinia virus and avian pox (fowlpox, pigeonpox, canarypox, etc.), adenoviruses, etc.

The invention therefore also relates to expression cassettes comprising such a DNA fragment and the means for regulating expression in the chosen expression system. It also relates to the expression system or expression vector, comprising such an expression cassette, in particular plasmid, poxvirus, adenovirus, as seen above.

Finally, the subject of the invention is the use of phages expressing the peptide of interest or a combination of phages expressing the peptides of interest as a diagnostic reagent, as an antigenic component or vaccine.

The invention also relates to the use of at least one peptide in accordance with the invention in combination or not with at least one recombinant vector in accordance with the invention for the preparation of a pharmaceutical composition intended for preventing or curing a condition related to the HIV virus. A composition according to the invention can comprise preparations which can be in the form of creams, lyophilized powders or not, solutions, suspensions, for administrations by mucous route such as oral, nasal, rectal, genital, cutaneous, for example. For parenteral administration such as intra dermal, subcutaneous, intramuscular, intravenous, intra arterial, intra lymphatic or intra peritoneal, for example, sterile injectable preparations may be in the form of solutions, suspensions or emulsions . In addition to the active ingredient (s), in accordance with the subject of the invention, the preparations may contain excipients and / or stabilizing agents suitable for the mode of administration.

Preparations for vaccine use may also contain adjuvants or be incorporated into delivery systems compatible with use in human medicine. We can report in particular the use of adjuvants such as Alum (aluminum phosphate phosphate or aluminum hydroxide or the mixture of the two) incorporated in a conventional manner in vaccines, the incomplete Freund's adjuvant, monophosphorylated lipid A ( MPL), QS21, Polyphosphazene, muramyl dipeptide (MDP) or its derivatives, the use of antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62, SB 26), ISCOMS, liposomes, microspheres composed of PLGA polymers of well calibrated diameter, or possibly pseudo virions.

The doses and routes of administration of these pharmaceutical compositions will be determined taking into account the nature of the composition, the level of expression of the peptide of interest by the recombinant vector if it is included in the preparation, the age , sex and weight of the individual receiving the preparation. The relative importance of the carrier molecule in the conjugate will also be taken into account if it is included. in the composition.

Given all of these factors which are known and appreciated by those skilled in the art, the doses of peptides administered may reach 1 to 5 mg but more generally will be between 5 μg and 1 mg per injection, preferably 50 to 500 μg. The recombinant vector coding for the peptide of interest may be administered or used to transfect or infect the cells of interest at a minimum dose of 10 ³ ′ ⁵ infecting units (pfu or plaque forming unit). Preferably, the recombinant vector will be used in a dose scale ranging from 10 ⁴ to 10 ¹⁰ pfu depending on the expression efficiency of the peptide by this vector and in particular in a dose scale ranging from 10 ⁶ to 10 ⁹ pfu, for example. When the pharmaceutical composition comprises several recombinant vectors coding for peptides of different interest, it is understood that these same dose scales can be applied to these combinations. Those skilled in the art will be able to refer to the protocols and clinical trials using preparations based on recombinant vectors, in particular recombinant pox viruses, recombinant adenoviruses, already produced in humans to agree on the appropriate number of pfu that must contain pharmaceutical composition.

When the pharmaceutical composition comprises a plasmid containing the expression system of the peptide of interest, it will be taken into account in the assay of this composition the level of immune response that this composition is capable of inducing which must be at least equal to that of. intact or modified peptide and / or the level of expression of the peptide induced by the plasmid in the cells of the organism which must approach as much as possible that obtained by the recombinant vectors already mentioned. For example, the quantities of plasmids contained in the pharmaceutical compositions may be in scales ranging from 1 μg to 100 mg, preferably between 0.1 mg to 10 mg. Those skilled in the art will be able to refer to the protocols and clinical trials already carried out in humans, using plasmid DNA preparations to agree on the dose of plasmid which the pharmaceutical composition must contain. For the prevention of HIV infection, the pharmaceutical composition may be administered at once or several times to achieve the desired level of response including in particular the level and quality of the specific antibody response and / or cell-specific mediation desired, and characterized in that it guarantees the protection of the individual against accidental contamination. To achieve this objective, it may be necessary, in addition to the composition of the preparation, the chosen administration voice, to respect the time limits set between each injection, which can preferably be 1 month, 2 months or 6 months and / or to make use, in a combined or alternating manner, during the duration of the medical protocol, in particular the vaccine protocol, of different pharmaceutical compositions relating to the peptide, to the recombinant vector, to the plasmid of interest or even to the phages expressing the peptide (s) of interest that the the skilled person is capable of mastering. To maintain the level of protection, it may also be necessary to give booster injections at regular intervals.

For the treatment of HIV-related infection, the pharmaceutical composition, in particular the vaccine composition, may be administered at once or several times and in a manner which may be very close together, in particular within periods of less than a week, in order to reach the level desired response, in particular that which makes it possible to ascertain the absence of the HIV virus in the blood by the PCR test. If necessary, the pharmaceutical composition comprising the peptide, the vector, the plasmid of interest. Or even phages expressing the peptide or peptides of interest may be combined or used alternately with conventional treatments for this condition, including in particular mono , bi or tri antiviral therapy.

Whether for the prevention or treatment of HIV infection, it may also be useful to use a pharmaceutical composition comprising one or more peptides of interest, the corresponding recombinant vector (s) as well as the plasmid (s) of interest or even bacteriophages expressing the peptide (s) of interest to stimulate the cells of the patient's immune system in vitro or ex vivo and to then re-inject them into the body of the individual. This methodology was notably developed in the immunotherapeutic treatment of cancer.

The subject of the invention is finally the use of the peptides of interest as a reagent for the diagnosis of HIV infection, making it possible in particular to identify subjects more resistant to infection also called "Long term non progressor" or conversely, to identify infected subjects more likely to develop AIDS quickly. For the first time, new conformational epitopes of the HIV envelope have been defined.

These peptides can therefore be used, for diagnostic purposes, to preferentially search for neutralizing antibodies to primary isolates of HIV, which very often recognize conformational epitopes and thus make it possible to distinguish individuals "Long term non progressor" (possessing antibodies neutralizers) of those who are likely to progress quickly to AIDS if no treatment, in particular anti-viral, is put in place quickly (not having neutralizing antibodies).

The present invention therefore also relates to a method of diagnosing HIV infection and / or of the susceptibility of infected subjects to develop AIDS quickly, the said method being preferably based on the analysis of the humoral response. For the analysis of the humoral response, immunoenzymatic, radioimmunological or western blotting methods well known to those skilled in the art may be used, such as, for example, the ELISA, RIA, RIPA or IRMA methods.

Description of the figures

Figures 1 to 5 show, depending on their dilution, the phage binding curves expressing respectively the peptide sequences SEQ ID

NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NAME 1 to a recombinant antibody specific for Ggp 160 and derived from the combinatorial library of antibodies made from lymphocytes d 'a subject "long term non progressor" (B) and an IgG antibody not specific for HIV envelope antigen (•), these two antibodies being previously fixed on ELISA plates. The intensity of the fixation is proportional to the value of the optical density (OD) obtained by ELISA.

The present invention is described in more detail below with the aid of the additional description which follows, which refers to examples of selection of antibodies directed against the HIV envelope, of selection of peptides according to the invention, synthesis of peptides according to the invention, induction of antibodies specific to peptides according to the invention, vaccine compositions according to the invention and use of peptides according to the invention for the diagnosis of HIV infection. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention of which they do not in any way constitute a limitation.

EXAMPLE 1 Selection of Peptides

The manufacture of recombinant antibodies using molecular biology methods have been widely developed over the past ten years and are now well known to those skilled in the art. It is also known that the specificity of a recombinant antibody is carried essentially by the CDR3s of the light and heavy chains. Knowledge of the chain of amino acids which represents CDR3 and of the skeleton structure of the heavy and light chains of a given antibody is sufficient for those skilled in the art to be able to reproduce and reconstitute an equivalent recombinant antibody having the same characteristics. of recognition of said antibody.

The sequences coding for the heavy and light chain parts of the selected Fab molecules can be isolated and synthesized, and cloned in any vector or replicon allowing their expression.

Any suitable expression system can be used, for example bacteria, yeasts, insect, amphibian and mammalian cells. Expression systems in the bacteria include those described in Chang et al. (1978) Nature 275: 615, Goeddel et al. (1979) Nature 281: 544, Goeddel et al. (1980) Nucleic Acids Res. 8: 4057, EP-A-36,776, US-A-4,551, 433, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80: 21-25, and Siebenlist et al. (1980) Cell 20: 269. The expression systems in yeasts include those described in Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1929, Ito et al. (1983) J. Bacteriol. 153: 163, Kurtz et al.

(1986) Mol. Cell. Biol. 6: 142, Kunze et al. (1985) J. Basic Microbiol. 25: 141,

Gleeson et al. (1986) J. Gen. Microbiol. 132: 3459, Roggenkamp et al. (1986) Mol.

Gen. Broom. 202: 302, Das et al. (1984) J. Bacteriol. 158: 1165, De Louvencourt et al. (1983) J. Bacteriol. 154: 737, Van den Berg et al. (1990) Bio / Technology 8:

135, Kunze et al. (1985) J. Basic Microbiol. 25: 141, Cregg et al. (1985) Mol. Cell.

Biol. 5: 3376, US-A- 4,837,148 and 4,929,555, Beach et al. (1981) Nature 300: 706,

Davidow et al. (1985) Curr. Broom. 10: 380, Gaillardin et al. (1985) Curr. Broom. 10:

49, Ballance et al. (1983) Biochem. Biophys. Res. Common. 112: 284-289, Tilburn et al. (1983) Gene 26: 205-221, Yelton et al. (1984) Proc. Natl. Acad. Sci. USA 81:

1470-1474, Kelly et al. (1985) EMBO J. 4: 475479; EP-A-244,234 and WO-A-

91/00357 .. The expression of heterologous genes in insects can be carried out as described in US-A-4,745,051, EP-A-127,839 and EP-A-155,476, Vlak et al.

(1988) J. Gen. Virol. 69: 765-776, Miller et al. (1988) Ann. Rev. Microbiol. 42: 177, Carbonell et al. (1988) Gene 73: 409, Maeda et al. (1985) Nature 315: 592-594,

Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8: 3129, Smith et al. (1985) Proc.

Natl. Acad. Sci. USA 82: 8404, Miyajima et al. (1987) Gene 58: 273, and Martin et al.

(1988) DNA 7: 99. Many strains and variants of baculovirus and permissive insect cells are described in Luckow et al. (1988) Bio / Technology 6: 47-55, Miller et al. (1986) GENERIC ENGINEERING, Setlow, J.K. et al. Eds. Flight. 8

Plenum Publishing, pp. 277-279, and Maeda et al. (1985) Nature 315: 592-594.

Expression in mammalian cells can be achieved as described in

Dijkema et al. (1985) EMBO J. 4: 761, Gorman et al. (1982) Proc. Natl. Acad. Sci.

USA 79: 6777, Boshart et al. (1985) Cell 41: 521, and US-A-4,399,216. We can also refer to Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980) Anal.

Biochem. 102: 255, US-A-4,767,704, 4,657,866; 4,927,762; 4,560,655; US patent

RE 30,985, WO-A-90/103430 and WO-A-87/00195.

Peripheral blood lymphocytes from a subject infected with the HIV virus and belonging to the group of "long-term non progressors" are used to produce the combinatorial library of antibodies. The lymphocyte cDNA is obtained from RNA using a methodology developed by Sodoyer R. et al. (1997) Human Antibodies 8: 37. The library of heavy and light chains is constructed using the phagemids pVH (pM 831) and pVL (pM452). The two libraries are then associated in a "Random" fashion by sub-cloning of the VL genes in the heavy chain library. The phagemid library obtained is then infected with the phage helper M13 VCS, thus allowing the expression of Fabs on the surface of the phages. After selection of the phages expressing the Fabs on their surface by "panning" against the Gp 160 protein, the nucleotide sequence of the recombinant Fabs expressed by the positive isolates and in particular the CDR3 sequences carried by the fragments of heavy and light chains is determined.

One of the recombinant antibodies specific for Gp 160 originating from this combinatorial library is then used to select the peptide sequences SEQ NOM to SEQ NOM 1 from a commercially available phage library expressing peptides randomly (pHD7, NEB). operating as follows: 1, 4 10 ¹¹ phages are incubated with 30 or 300 ng of the recombinant antibody according to example 1 in 200 μl of PBS-0.1% tween 20 for 20 min at 20 ° C. The mixture is transferred to a tube containing 50 μl of protein G coupled to sepharose beads previously balanced for 1 hour in 1 ml of PBS-0.1% tween 20 containing 5% skimmed milk. After a further 20 min incubation, the beads are centrifuged and washed 3 times with 1 ml of PBS-0.1% tween 20. Au

4 ^th wash, the beads are times with PBS-0.1% tween 20 containing 5% skimmed milk, incubated 10 min, centrifuged and rinsed again three times with PBS

0.1% tween 20. After the last centrifugation, the beads are taken up in 1 ml of 0.2 M glycine-HCl, pH = 2.2, incubated for 10 min at 20 ° C. and centrifuged. The supernatant is transferred to a tube containing 60 μl of Tris base 2M pH = 7.5, so as to neutralize the solution and then incubated with 2 ml of E. Coli 7118 bacteria in the exponential growth phase for 15 min. The culture volume was made up to 100 ml with LB medium, and the incubation was continued for 4 h at 37 ° C. After a centrifugation intended to eliminate the bacteria, the phages are precipitated by adding 25 ml of PEG 20% -NaCI 2.5M in the culture supernatant overnight at 4 ° C. After centrifugation (10,000 rpm, 20 min, 4 ° C), the phage residue is taken up in 1 ml of PBS-0.1% tween 20-1% skimmed milk, and titrated. Screening is complete when the whole process has been repeated 3 to 4 times. To complete the selection of phages expressing peptide sequences 1 to 11, they were also tested by ELISA as follows: 0.2 mg of recombinant antibody specific for Gp 160 or of an antibody which does not have specificity for an antigen of the envelope of HIV called "control Ig", diluted in 50 μl of PBS, is deposited in each well of an ELISA plate followed by an incubation overnight at 4 ° C. After replacing the antibody solution with 0.1 ml of PBS-0.1% tween 20 (PBST) containing 5% skimmed milk and incubation for 1 hour at 37 ° C, and carrying out a dilution range of reason 2 on the phages expressing the peptide sequences, SEQ ID NOM to SEQ ID NOM 1 in PBST-1% skimmed milk, the various dilutions made are distributed in the wells sensitized either by the specific recombinant antibody according to Example 1, or by the control Ig. After 2 h at 37 ° C., the phages are removed by aspiration of the dilutions then the wells washed 10 times with 0.2 ml of PBST. The wells are then incubated for 1 hour at 37 ° C. with a 1 000 000 solution in PBST-1% milk of a biotinylated antibody directed against the phage fd (SIGMA). After a new series of washes in PBST-1% milk, a streptavidin-peroxidase complex (SIGMA) diluted 1: 2000 in PBST is added to each well followed by an incubation of 1 h at room temperature and washes in PBST- 1% milk. The enzymatic activity of peroxidase is conventionally revealed by the addition of a solution of OPD diluted to 1 mg / ml in sodium citrate buffer. The intensity of coloration of the OPD solution is then measured with a spectrophotometer, then the OD curves (optical density) are established as a function of the dilution of phages. The curves in FIGS. 1 to 5 show, by way of example, that the phages expressing the peptide sequences, SEQ ID NO: 7 to SEQ ID NO: 11 bind well to the specific recombinant antibody (OD> 0.6 observed for at least one dilution of phages) while there is no significant fixation on the control Ig (OD <0.3 whatever the dilution of phages tested in the dilution range going from 5 10 ¹¹ phages / well at 4.8 10 ⁸ phages / well). The positive phages, ie those which bind specifically to the specific recombinant antibody, are amplified in E. Coli. Mini phage DNA preparations are carried out according to the procedures described in the works of Maniatis, the DNA is sequenced using an automatic sequencer from which the sequence of the peptide expressed by the phage is deduced. Example 2 Synthesis of Peptides

The peptides from SEQ ID NOM to SEQ ID NOM 1, are synthesized in solid phase by referring to the methodologies developed in the works Solid phase peptide synthesis: a practical approach, IRL Press, Oxford, 1989 and Solid phase peptide synthesis, second edition, published by Pierce Chemical Company,

1984.

The α amino function of the amino acids is protected by the introduction of a t-butyloxycarbonyl (t-boc) group, thus allowing coupling, by the carboxylic function of the amino acid, to an active chloromethylee resin. After fixation on the resin, the amino function is "deprotected" by the action of trifluoroacetic acid followed by a neutralization step with triethylamine. The amino function thus released then undergoes a coupling reaction with another amino acid in the form of a t-boc derivative via carbodiimides. This process is implemented by the ABI automaton (Applied Biosystem Inc) 430A which thus performs the automatic synthesis of peptides. At the end of the synthesis, the peptide is detached from the resin by the action of hydrofluoric acid. The extract is then purified by reverse phase HPLC using a Vydac C4 type semi-preparative column and an acetonitrile gradient ranging from 15 to 55% in a 0.1% trifuoroacetic acid solution. Liquid chromatography is programmed for a period of 40 min with a flow rate of 2 ml / min. The purity of the peptides is controlled by analytical chromatography and exceeds 95%.

Example 3 Induction of Specific Antibodies

1) Induction of antibodies in guinea pigs and rabbits

The peptides of SEQ ID NOs: 1 to 6, displayed on phage are injected into guinea pigs and rabbits: two injections of 100 microliters intravenously, 3 weeks apart followed by a final bleeding 15 days after the 2 ^ee injection. The sera are tested in ELISA against gp160 and a reaction against the glycoprotein is observed. These peptides are therefore capable of inducing a response against gp160. 2) Induction of antibodies in mice

The various phage isolates expressing the peptides defined by SEQ ID NO: 7 to SEQ ID NOM 1 are purified on a cesium chloride gradient. 5 groups of BaLB / c mice are then identified. Each group is immunized intraperitoneally, 3 times at 3 week intervals with a single type of purified isolate expressing either SEQ ID NO: 7, or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NOM0 or SEQ ID NOM 1 at the rate of 10 ¹² phages purified by injection. To compare the antibody responses, we introduced 2 additional groups of mice, the first group receiving 3 injections of 10 ¹² phages expressing peptides not mimicking conformational epitopes of the HIV envelope (irrelevant phages), the second receiving 3 injections of 5 μg of Gp160 protein. We also introduced an 8 ^th group of mice which received a mixture of phage isolates, comprising phages expressing in equal parts SEQ ID NO: 7,

SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NOM0 and SEQ ID NOM 1. Some mice in this group as well as some of the mouse group immunized with the unrelated phages received ^4th injection of 5 mcg Gp 160, 3 weeks after the 3 ^rd injection. The analysis of the specific antibody response is carried out on the serum of the mice of each group taken 15 days after each injection. Analysis of the specific antibody response includes the detection of anti gp 160 antibodies by ELISA using plates sensitized with the Gp 160 protein using a procedure similar to that described in example 1 and the search for antibodies. neutralizers. For the search for neutralizing antibodies, the dilution of serum is determined which prevents the formation of syncytia in 50% of the microwells infected with

10 CCID50 of a strain of HIV virus. After decomplementation of the sera and production of a dilution range of reason 2 in RPMI medium, 500 μl of the suspension of HIV virus titrating 10 ^2.5 CCID ₅₀ / ml is mixed with 500 μl of the various dilutions of sera. After an incubation of 2 hours at 37 ° C, the mixture is deposited in a volume of 100 μl on CEMss cells previously fixed in microwells (6 microwells / dilution of serum). After 1 hour of contact at 37 ° C. with the CEMss cells, the mixture is aspirated and replaced with culture medium. After 7 and 14 days of incubation, the cultures are examined under a microscope for the enumeration of the syncytia. The neutralizing titer of 50% is determined according to the Spearman and Kârber method. A good production of antibodies is observed in the mice of the 8 ^th group which has been immunized with the mixture of phage isolates expressing the different peptide sequences (SEQ ID NO: 7 to SEQ ID NOM 1)

EXAMPLE 4 Vaccine Formulations

A peptide having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 and produced by chemical synthesis according to Example 2 or obtained from the expression product of a recombinant vector and in particular of a recombinant baculovirus using the techniques developed by Smith et al (USA 4,745,051). Water-in-oil emulsions are then prepared using squalene as a constituent of the organic phase, tween 80 or a mixture of tween 80 and SPAN as a surfactant, the aqueous phase containing the peptide solution. When the hydophobicity of the peptide is very high, oil-in-water emulsions are produced in which the peptide will be associated with the organic phase. If necessary, immunostimulants such as QS 21, derivatives of MPL, or any other adjuvant are incorporated in the preparation of these emulsions. This formulation is used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

EXAMPLE 5 Vaccine Formulations

A vaccine formulation based on liposomes is prepared comprising a peptide having one of the sequences SEQ ID NOM to SEQ ID NO: 11 described in the example

1 and produced by chemical synthesis according to Example 2 with reference to works such as "Liposomes as Drug Carriers" published by G. Gregoriadis, 1988, or to volumes 1 to 3 of "Liposome Technology edited by G. Gregoriadis, 1984. This formulation is used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

EXAMPLE 6 Vaccine Formulations

A vaccine formulation based on ISCOMs is prepared containing a peptide having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 and produced by chemical synthesis according to Example 2 with reference to B Morein et al, 1984, Nature 308: 457 or B Morein et al Immunoiogy toDay, 1987, 8 (11): 333.

EXAMPLE 7 Vaccine Formulations

A formulation based on micro particles is prepared comprising a peptide having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in the example

1 and produced by chemical synthesis according to Example 2 mimicking a conformational epitope of the envelope of HIV. For the preparation of micro particles or nanoparticles, many synthetic or natural polymers are used such as the polymer of methyl metacrylate (Troster SD et al, 1992, J. Microencaps. 9:19) but often poly (d, 1 -lactide- co-glycolide) also called PLGA is the referent because of its biodegradability, its safety and its applications already old in the medical field. The PLGA micro particles loaded with peptides are prepared in particular by double water-in-oil-in-water emulsion. The peptide is dissolved in the aqueous phase and then emulsified in a solution of PLGA in the organic phase such as dichloromethane. The water in oil emulsion is obtained by high speed stirring of the peptide solution in the organic solution of PLGA. Then a second aqueous phase containing an appropriate concentration of surfactant such as polyvinyl alcohol is added to the first emulsion to thereby produce the double emulsion. Other surfactants are also used such as bile salts or poly (oxyethylene glycerol monoleate) to stabilize the double emulsion (Rafati H et al., 1997, Vaccine 15: 1888). After stirring overnight to allow the solvent to evaporate, the microparticles of PLGA are washed several times in distilled water and then lyophilized and kept at 5 ° C. This formulation is used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 8: Vaccine composition

Peptides with fewer than 20 amino acids can be weakly immunogenic. To increase the immunogenicity of peptides having any of SEQ ID NOM to SEQ ID NOM 1 sequences described in Example 1 and produced by chemical synthesis according to Example 2, a vaccine formulation is prepared based on polymers of the same peptide or different peptides, in the form of octamers comprising a branched poly-lysine structure with 8 lateral arms on which the same peptide or different peptides are fixed according to Example 1 by implementing the method developed by Posnett DN et al. (1988) J. Biol. Chem. 263: 1719. This formulation is used for the preparation of a vaccine composition for the prevention or treatment of HIV virus infection.

Example 9: Vaccine composition

A vaccine composition is produced comprising a peptide having one of the sequences SEQ ID NAME, SEQ ID NO: 3 or SEQ ID NO: 4 described in Example 1 and produced by chemical synthesis according to Example 2, according to Example 2 framed by the 2 T helper P24E and T epitopes of the HIV virus, these 2 epitopes playing the role of carrier molecule and thus strengthening the immunogenicity of the peptide. The coupling reaction of the peptide with these 2 T helper epitopes is carried out in 2 stages according to conventional methods well known to those skilled in the art. Firstly, the N-terminal part of the peptide is coupled with the C-terminal part of the peptide sequence representing the p24E epitope by intercalating a space consisting of the glycine-praline-glycine sequence. Then, in a second step, the terminal C part of the intermediate product is coupled with the N terminal part of the peptide sequence representing the T1 epitope by intercalating the same glycine-proline-glycine sequence to obtain the final product. This formulation is used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 10: Vaccine composition comprising a lipopeptide

A vaccine formulation is prepared comprising a peptide having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 and produced by chemical synthesis according to Example 2 coupled to one or more chains derived from fatty acids, including N _ε palmitoyl-lysine, N, N-dipalmitoyl-lysine, pimelautide, trimexautide or to a steroid group, including Nε [(cholest-5-enyl-3-oxy) -acetyl)] - iysine or (cholest-5-enyl-3-oxy) acetic acid according to the process described in patent EP0491628 (INSERM) so as to obtain a lipopeptide. This formulation is used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 11 Expression of Peptides by Poxviruses

A vaccine composition is prepared comprising a recombinant poxvirus encoding a peptide having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 mimicking a conformational epitope of the envelope of HIV. Recombinant poxviruses are obtained by homologous recombination from embryonic chicken cells infected with poxviruses and co-transfected with plasmids containing an expression cassette, flanked at the ends of DNA sequences homologous to those of nonessential regions of l Poxvirus DNA, and containing, under the dependence of poxvirus promoters (H6, 13L), the poly nucleotide which codes for the peptide according to Example 2 using the methods described in US Patents 4,769,330, 4,772,848, 4,603,112, 5,100,587 ,

5,179,993 and 5,863,542. These recombinant poxviruses are used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 12 Expression of Several Peptides by a Poxvirus

A vaccine composition is prepared comprising a recombinant poxvirus encoding several peptides having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 mimicking several conformational epitopes of the envelope of HIV. The use and preparation of recombinant vectors coding for several epitopes is well known to those skilled in the art (Toes RE et al. (1997) Proc. Natl. Acad. Sci. USA 94: 14660, Thomson SA et al. (1996) J. Immunol. 157: 822) and is also applicable to the preparation of recombinant poxviruses encoding multiple mimotopes. In particular, a vaccine composition is prepared comprising a recombinant canaripox (recombinant ALVAC) encoding multiple mimotopes of the HIV envelope. These recombinant poxviruses are used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 13 Combinations of Peptides

A vaccine composition is prepared comprising several peptides having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in example 1 using the different preparation methods described in examples 2 to 12 mimicking several conformational epitopes of the envelope of HIV. These different compositions are used for the preparation of a vaccine composition intended for the prevention or treatment of HIV virus infection.

Example 13: Diagnosis

HIV specific antibody detection is carried out by ELISA using one or more peptides having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 and produced by chemical synthesis according to Example 2 for the diagnosis of HIV infection from a biological sample In general, a sample of physiological fluid (blood, plasma, serum) is taken, a sample which is then reacted in the presence of a peptide according to the invention.

To do this, the peptide itself is used as a diagnostic reagent. Generally, either an indirect diagnostic test, of the ELISA type, in which the peptide fixed on a support (well) is placed in the presence of the sample to be tested, while the revelation of the antigen-antibody binding is ensured by a labeled anti-Ig, either by a competition or displacement test, in which a peptide according to the invention is used, and a labeled antibody specific for the peptide. The peptide is there also fixed to a solid support such as well, strips. In the competition test, the peptide is placed simultaneously in the presence of the sample

(sample antibody) and a labeled antibody specific for the peptide. Antibodies labeled with peroxidase are generally used. In the competition or displacement test, monoclonal and polyclonal antibodies or recombinant antibodies specific for the peptide according to the invention are generally used, which are sometimes Fab or F (ab) ₂ fragments and in particular those described in the invention.

Example 14: Diagnosis

HIV specific antibody detection is implemented by immunochromatography using one or more peptides according to the invention for the diagnosis of HIV infection from a biological sample In this case the peptide according to the invention is fixed on a strip-type support and reference is made to the article by Robert FN Zurk et al., Clin. Chem. 7/31, 1144-1150 (1985) and in patents or patent applications WO-A-88/08 534, WO-A-

91/12528, EP-A-291 176, EP-A-299428, EP-A-291 194, EP-A-284 232, US-A-5 120 643, US-A-5 030 558, US-A -5 266 497, US-A-4 740 468, US-A-5 266 497, US-A-4 855 240, US-A-5 451 504, US-A-5 141 850, US-A-5 232 835 and US-A-5 238 652 for implementing the method.

Example 15: Diagnosis

Study of the specific lymphoproliferative response to one or more peptides according to the invention for the diagnosis of HIV virus infection from a biological sample.

The patient's blood is collected on a heparin tube. The lymphocytes are then separated by centrifugation on hypaque Ficoll and then distributed in 96-well sterile microplates at the rate of 2 × 10 ⁵ cells per round-bottom well under a final volume of 200 μl of complete culture medium (RPMI 1640 supplemented with 25 mM HEPES, 2 mM L -glutamine, 50U / ml of penicillin, 50μg / ml of streptomycin and 5% serum AB decomplemented) and placed in the presence of variable concentrations of one or more peptides having one of the sequences SEQ ID NOM to SEQ ID NOM 1 described in Example 1 and produced by chemical synthesis according to Example 2 (concentrations ranging from 1ng / ml to 50μg / ml). Each concentration of peptide is tested in triplicate to overcome biological variations as well as possible. Multiple combinations of peptides can also be tested in the indicated concentration range, for example a combination resulting from the association of an envelope mimotope with a nucleocapsid mimotope in the indicated concentration range. After 5 days of culture at 37 ° C. under 5% CO2, 0.5 μci of tritiated thymidine is added to each well. After a further 16 hour incubation, the cellular DNA is collected from each culture well on filters after precipitation with ethanol and the incorporation rate of tritiated thymidine is measured using a liquid scintillation counter. which reflects the intensity of the lymphoproliferative response. The results are expressed in the form of stimulation index (average of the cpm of the lymphocyte culture wells containing a given concentration of peptide / average of the cpm of the lymphocyte culture wells without peptide). The lymphoproliferative response is considered positive when the stimulation index is greater than 3.

Claims

1) Peptide for the prevention or the therapeutic treatment of the infection with HIV virus capable of interacting with an antibody specific for an antigen of the envelope of said virus and coming from an HIV positive patient and belonging to the group of "long term non progressor ", comprising an amino acid sequence which mimics a conformational epitope of an antigen of said envelope without however corresponding to a continuous amino acid sequence of this antigen.

2) Peptide according to claim 1, according to which the envelope antigen is represented by the envelope protein gp160.

3) Peptide according to claim 1 or 2, characterized in that this peptide can comprise the sequences 1 to 11 SEQ ID NO: 1 Phe Asn Leu Thr His Phe Leu

SEQ ID NO: 2 Glu Gly Trp His Ala His Thr

SEQ ID NO: 3 Lys Leu Asn Trp Met Phe Thr

SEQ ID NO: 4 Ser Thr Asn Trp Met Phe Thr

SEQ ID NO: 5 Ala Met Pro Leu Pro Tyr Thr Phe SEQ ID NO: 6 Asp Ser His Thr Pro Gin Arg

SEQ ID NO: 7 Val Ser Phe Thr Pro Ser Phe

SEQ ID NO: 8 His Ala Ala Leu Ser Met Asn Thr His Ala Leu Met

SEQ ID NO: 9 Ala Trp His Glu Ser Arg Ala

SEQ ID NO: 10 Phe Lys Thr Ala Tyr Pro Thr SEQ ID NO: 11 Ser His Ala Leu Pro Leu Thr Trp Ser Thr Ala Ala

4) Peptide comprising the chain of at least two peptides according to one of claims 1 to 3.

5) Peptide according to claim 4 comprising the duplication of identical peptides.

6) Peptide according to claim 5 wherein the coupling of the two peptides is done by means of a "spacer" arm consisting of the amino acid sequence Gly Pro Gly 7) Conjugate comprising at least one peptide according to one of claims 1 to 6 linked to a carrier molecule to induce or strengthen the immunogenicity of said peptide.

8) Conjugate according to claim 7 according to which the carrier molecule comprises at least one T helper epitope of the HIV virus.

9) Conjugate according to claim 8, the carrier molecule of which comprises the epitope p24E and T1 of the HIV virus

10) Conjugate according to claim 9, comprising a peptide resulting from the duplication of the NOM, NO: 3 or NO: 4 sequences according to claim 6, said peptide being linked on the N terminal side to the p24E epitope and on the C side to the T1 epitope via 2 “spacer” arms.

11) Conjugate according to claim 10, characterized in that the 2 spacer arms are identical and consist of the Gly Pro Gly sequence and that it optionally includes one of the sequences 12 to 14

SEQ ID NO: 12 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Gly Pro Gly Lys Leu Asn Trp Met Phe Thr Gly Pro Gly Lys Leu Asn

Trp Met Phe Thr Gly Pro Gly lys Gin Ile Ile Asn Met Trp Gin Glu Val Glu Lys Ala Met Tyr Ala

SEQ ID NO: 13 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Gly Pro Gly Ser Thr Asn Trp Met Phe Thr Gly Pro Gly Ser Thr Asn

SEQ ID NO: 14 Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Gly Pro Gly Phe Asn Leu Thr His Phe Leu Gly Pro Gly Phe Asn Leu

Thr His Phe Leu Gly Pro Gly Lys Gin Ile Ile Asn Met Trp Gin Glu Val Glu Lys Ala Met Tyr Ala 12) Recombinant vector comprising a functional expression cassette allowing the expression of a poly nucleotide coding for a peptide according to one of claims 1 to 11

13) Recombinant vector according to claim 12, characterized in that it is an adenovirus, a poxvirus, a baculovirus, a bacteriophage or a plasmid.

14) Therapeutic or prophylactic composition for HIV infection, in particular intended for vaccine use, the active principle of which comprises a peptide according to one of claims 1 to 11 and / or a recombinant vector coding for said peptide according to one of claims 12 and 13.

15) Composition according to claim 14, the active principle of which is in the form of a formulation associated with an adjuvant compatible for the administration of an effective dose by mucous or parenteral route.

16) Use of a peptide according to one of claims 1 to 11 and / or of a recombinant vector according to one of claims 12 and 13 as a reagent for the diagnosis of HIV, said diagnosis comprising evaluation, from a blood sample, from the humoral and / or cell-mediated response specific for this peptide.

77,) Use of a peptide according to one of claims 1 to 11 and / or of a recombinant vector according to one of claims 12 and 13 as a reagent for the diagnosis of the susceptibility of subjects infected with the virus HIV, to rapidly develop AIDS.

18) Use of a peptide according to one of claims 1 to 11 and / or of a recombinant vector according to one of claims 12 and 13 for the preparation of a therapeutic or prophylactic composition, intended for the treatment or prevention of HIV infection.

19) Use of a peptide according to one of claims 1 to 11 and / or of a recombinant vector according to one of claims 12 and 13 to stimulate cells of the immune system of an individual in vitro, said cells being then intended to be reinjected into the body of the individual after stimulation.