FR2830534A1 - New polypeptide based on human immune deficiency virus gp41, useful in preventative or therapeutic vaccine, induces neutralizing antibodies against primary isolates - Google Patents

Abstract

Polypeptides (I) containing fragments of the HIV gp41 protein and able to adopt a alpha -helical conformation are new. Polypeptides of formula (I) containing fragments of the HIV gp41 protein and able to adopt a alpha -helical conformation are new, where (N-S1)n-C-(S2-N)m (I) N = amino acids (aa) 30-77 of gp41; C = aa 117-154 of gp41; S1 and S2 = aa sequence such that (I) adopts an alpha -helical conformation, or may be absent; n and m = 0-1, but not both 0 The alpha -helical conformation is determined by the program SOPMA using the parameters: number of conformational states = 4; limit of similarity = 8 and width of window = 70. Independent claims are also included for the following: (1) Conjugate (II) of (I) with a carrier protein or peptide; (2) DNA sequence (III) that encodes (I) or (II); (3) Vector that contains (III); (4) Host cell containing the vector of (3); and (5) Preparation of polypeptides by expression from cells of (4).

Description

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 The present invention relates to a polypeptide antigen derived from the gp41 protein, as well as its use for immunization against HIV-related infections., This work has been co-funded by the ANRS.

 The development of an immunization method against HIV is now one of the priorities of scientific research.

 The major obstacles of the high genetic variability of the virus and the low immune system exposure of neutralizing viral epitopes significantly hamper the development of neutralizing immunity.

 The HIV envelope glycoprotein that is required to confer the virus's infectivity represents the target of neutralizing antibodies. These characteristics have made the latter a subject of intense investigation.

 The glycoprotein envelope (env) of the human immunodeficiency virus-1 (HIV-1) is synthesized from the precursor gp160 which gives under the action of a protease the gp120 and gp41 subunits.

 The binding of gp120 / gp41 to cell receptors induces a conformational change in gp41 from a latent (non-fusogenic) state to a fusion-active (fusogenic) state.

Between these two states, there exists a so-called "intermediate" transient state during which gp41 is presented as a membrane protein in both the viral and cellular membranes (Weissenhorn et al Nature (1997), 387 (6631), 426-30). .

 The vaccine use of gp41 in its fusogenic conformation is described in WO00 / 40616. According to this application, the N helices can be used alone or in combination with the C helices to reproduce in the latter case the fusogenic conformation of the gp41.

 Liaison experiments have also established that the nonfusogenic latent state is characterized by the inaccessibility of large portions of the ectodomain of gp41. The gp120 interacts in effect so as to mask the epitopes. It has also been shown that the inhibition of the change of structure from the intermediate state to the fusogenic state by peptides used as competitors could affect the viral infection (Weissenhom W. et al, Molecular Membrane Biology, 1999 , 16, 3-9).

 The Applicant proposes a new polypeptide antigen that can be used for therapeutic and prophylactic immunization against HIV-related infections. The Applicant has indeed demonstrated for the first time that a polypeptide mimicking

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dans laquelle : N représente la séquence des acides aminés 30 à 77 de la gp41, C représente la séquence des acides aminés 117 à 154 de la gp41, SI et S2 sont indépendamment l'un de l'autre soit absents, soit représentent une séquence d'acides aminés telle que la séquence de formule 1 adopte une conformation en hélice

alpha telle que déterminée par le logiciel SOPMA dans les conditions suivantes : * nombre d'états conformationnels = 4 'Limite de similarité = 8 et, 'Largeur de la fenêtre = 70 n = 0 ou 1 ; m = 0 ou 1 et m + n = 1 ou 2. the intermediate state of gp41 is able to induce antibodies that neutralize primary HIV isolates
The present invention thus relates to a polypeptide comprising a sequence of formula 1:

in which: N represents the amino acid sequence 30 to 77 of gp41, C represents the amino acid sequence 117 to 154 of gp41, S1 and S2 are independently of each other either absent or represent a sequence of amino acids such that the sequence of formula 1 adopts a helical conformation

alpha as determined by the SOPMA software under the following conditions: * number of conformational states = 4 'Similarity limit = 8 and,' Width of the window = 70 n = 0 or 1; m = 0 or 1 and m + n = 1 or 2.

 Preferably, the polypeptide as defined above comprises a sequence of formula 1 in which m + n = 1.

 Preferably, SI is absent or represents the amino acid sequence D, DQ, DQQ, DQQL or DNNMT and S2 is absent or represents the amino acid sequence W, WA, WAS, WASL or WASLW.

 The amino acid sequences S1 and S2 are defined using the one-letter code in which D is aspartic acid, Q is glutamine, etc.

 According to a particular embodiment, the polypeptide comprises a sequence of formula 1 as defined above in which N represents SEQ ID. And C is SEQ ID Ni27.

 According to another embodiment, the polypeptide according to the invention comprises a further sequence of formula (G) aS- (H) b in which G represents a glycine residue, H represents a histidine residue, a is greater than or equal to 4 and b is greater than or equal to 6. Said sequence is linked by amide bond to the NH 2 -terminal or COOH-terminal end of the polypeptide according to the invention.

 According to another aspect, the present invention relates to a conjugate comprising a polypeptide according to the invention conjugated to a protein or a carrier peptide.

 According to another aspect, the present invention relates to a DNA sequence coding for a polypeptide according to the invention or for a conjugate according to the invention.

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 The present invention also relates to an expression vector comprising said DNA sequence as well as a host cell containing said vector.

 The present invention also relates to a pharmaceutical composition comprising at least one polypeptide as defined above, or at least one conjugate as defined above, or at least one expression vector as defined above, a pharmaceutically acceptable excipient and optionally an adjuvant.

 According to one particular embodiment, the present invention relates to a pharmaceutical composition as defined above that can be administered orally.

 The present invention therefore also relates to the polypeptide as defined above for its use as a medicament and in particular for its use for the immunization of the human body against HIV-related infections.

 Another object of the present invention relates to the process for preparing a polypeptide as defined above comprising the expression of said polypeptide from a host cell as defined above.

 The invention is described in more detail in the description which follows.

 The phenomenon of conformational change of gp41 preceding the fusion of cellular and viral membranes is illustrated in FIG. 1. The binding of gp120 on the receptor and the co-receptor of the virus causes a first modification which causes the unfolding of the peptide of fusion and its anchoring in the cell membrane. At this moment an intermediate state is formed in which there is no interaction between the helices N and C. The second event illustrated in FIG. 1 is the folding of the gp41 which corresponds to the adoption of a conformation. thermodynamically more stable of the molecule.

The energy released during this folding allows the approximation and fusion of the lipids of the cell and viral membranes.

 The applicant has surprisingly demonstrated that the polypeptide according to the invention which mimics the intermediate state of gp41 induces antibodies which neutralize the primary isolates of HIV. Induction of antibodies neutralizing the primary isolates can be readily determined by the neutralization assay as described in the article by C. Moog et al (AIDS Research and Human Retroviruses, Vol 13 (1), 13-27, 1997) which can be referred to for a complete description of the latter. In the context of the present invention it is believed that neutralizing antibodies were induced by the tested antigen when the 1/5 diluted serum resulted in a 10-fold reduction in the amount of p24 present in the culture supernatant.

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 This property makes the polypeptide according to the invention a vaccine candidate of interest for humans.

 The polypeptide according to the present invention has the particularity of adopting, under physiological conditions, a conformation that may be described as open in opposition to the closed conformation of gp41 in fusogenic form in which the N and C domains are paired. to each other in an anti-parallel orientation (Cf.

Fig. 1).

Indeed, the open conformation of the polypeptide according to the invention is characterized in that the C sequence is not matched to the N sequence in the anti-parallel orientation as present in the fusogenic form. The polypeptide according to the present invention is preferably in a trimeric form in which the N sequences and the C sequences are respectively paired with each other in a parallel orientation.

de sodium par DSC (calorimétrie différentielle à balayage) donne une valeur de 110 C, le début du phénomène de dénaturation apparaissant à environ 100 C à pH=2, 5. La thermostabilité de la protéine gp41 à pH neutre a été évaluée par Weissenhorn et al (EMBO, 1996,7, 1507-1514). Le Tm mesuré à pH neutre par ces auteurs est de 78 C, ce qui signifie que de façon surprenante cette protéine est plus stable à pH acide qu'à pH neutre. Ces résultats ont été confirmé par une analyse par dichroïsme circulaire visant à calculer le pourcentage d'hélice alpha dans la protéine. The Applicant has also surprisingly demonstrated that the polypeptide according to the invention retains its open conformation in a strongly acidic medium. This property makes the polypeptide according to the invention an orally administrable vaccine antigen. The Applicant has indeed shown that the ectodomain of the gp41 protein is extremely heat stable at pH2.5. The measurement of Tm (temperature at which 50% of the proteins present are denatured) of gp41 in 50 mM of formate

of sodium by DSC (differential scanning calorimetry) gives a value of 110 C, the beginning of the denaturation phenomenon appearing at about 100 C at pH = 2, 5. The thermostability of the protein gp41 at neutral pH was evaluated by Weissenhorn and al (EMBO, 1996, 7, 1507-1514). The Tm measured at neutral pH by these authors is 78 ° C., which means that, surprisingly, this protein is more stable at acid pH than at neutral pH. These results were confirmed by circular dichroism analysis to calculate the percentage of alpha helix in the protein.

The Applicant has also shown that the polypeptide according to the invention has the same peculiarity. This specific property makes the polypeptide according to the invention a vaccine antigen of choice for oral administration.

 This open conformation is obtained by the direct bonding of the N and C sequences as defined above. In such a case, SI and S2 are absent and the last amino acid of the N sequence is directly linked by amide bond to the first amino acid of the C sequence or vice versa (eg AA33-77-AA117-154). This open conformation is also obtained when the N and C sequences as defined above are connected to each other via a sequence S1 or S2 such that the resulting sequence of formula I adopts

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 an alpha helix conformation as determined by the SOPMA software under the following conditions: number of conformational states = 4; similarity limit = 8 and width of the window = 70.

 The open conformation according to the invention is characterized in fact by the absence of a flexible region between the sequences N and C. The SOPMA software available on the site: http: // npsa-pbil. IBCP. n / cgi-bin / npsaautomat. pl? page = / SNPA / npsasopma. html (GeourjonC and Deléage G. SOPMA, Casios (1995), 11, 681-684) makes it easy to determine whether the SI or S2 sequence adopts within the framework of the polypeptide of formula 1 an appropriate alpha-helix conformation in the context of the present invention. .

 According to a preferred embodiment m + n = 1 and if SI is absent S2 represents W, WA, WAS, WASL or WASLW and if S2 is absent SI represents D, DQ, DQQ, DQQL or DNNMT.

tel que défini ci-dessus les séquences AA30-AA77 et AA117-AA154 correspondent respectivement aux séquences SEQ ID N26 et SEQ ID Ni27. In addition, according to a particularly preferred embodiment, in the polypeptide

as defined above, the AA30-AA77 and AA117-AA154 sequences respectively correspond to the sequences SEQ ID N26 and SEQ ID Ni27.

 The AA30-AA77 and AA117-AA154 sequences resulting from gp41 are numbered, taking for reference the numbering of the sequence of the gp41 protein as given in the sequence listing under the heading SEQ ID? 25. The sequence SEQ ID? Corresponds to a truncated LAI gp41 protein in which the first amino acid A has the number 1.

 The AA30-AA77 and AA117-AA154 sequences of the present invention may be derived from any HIV 41 gp protein, including the HIV1 and HIV2 strains, including the laboratory strains and the primary isolates. Preferably, the sequences N and C according to the present invention are derived from an HIV1 strain and in particular from an HIV1 LAI strain.

 The nucleotide and peptide sequences of a large number of gp41 proteins are known and available, for example, on the Internet on the website http: // hiv-web. LANL. go as well as in the corresponding compendium of Los Adamos. It is clear that any sequence in which one or more conservative mutations that do not substantially alter either the immunogenicity or the open conformation has been introduced is also included within the scope of the present invention.

 The open conformation according to the present invention is characterized by a number of parameters measurable by the DSC technique and the circular dichroism technique. These techniques are described in detail in the following articles by A. Cooper and

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 al, Phil Trans. R. Soc. Lon. A (1993). 345,23-25, and V. V. Plotnikov et al. Analytical Biochemistry 250,237-244, (1997) and S. M. Kelly and N. C. Price (1997) Biochim. Biophys.

Acta. 1338.161 to 185.

DSC measures the thermodynamic parameters of protein denaturation. Denaturation of proteins is an endothermic event that can be evaluated by measuring the heat absorption by the protein as a function of temperature. The two main parameters obtained are: - Tm or half-denaturation point (ie temperature at which 50% of the protein is present in native form and 50% in denatured form) and - AH or enthalpy variation during denaturation (ie the heat needed to denature the protein).

These two parameters are precise markers of the conformation of a protein.

A DSC analysis is detailed in Example 5 with reference to Figure 2.

 The circular dichroism technique is used to evaluate the secondary structure of the protein. The signal of an alpha helix is characterized by minima at 208nm and 222nm. The signal value at 222nm is used to determine the percentage of alpha helix in a protein. The signal of a loop is characterized by a maximum at about 205 nm. This technique makes it possible to demonstrate that the polypeptide according to the invention does not contain the loop normally present in the fusogenic form and that it consists almost exclusively or exclusively of an alpha helix.

Circular dichroism analysis is detailed in Example 6 with reference to FIG.

 Although the polypeptide according to the present invention has an open conformation that is stable, this conformation can be enhanced by adding cysteine residues to the ends of the polypeptide. For this purpose, two additional cysteine residues may be added at the N-terminus or at the C-terminus, preferably at the N-terminus of the polypeptide according to the invention for covalently fixing the trimer in an open conformation.

 The polypeptide according to the invention can be obtained by any conventional technique of chemical synthesis or genetic engineering.

When the polypeptide is produced by chemical synthesis, the polypeptide according to the invention can be synthesized in the form of a single sequence, or in the form of several sequences which are then linked to each other. The chemical synthesis can be carried out in solid phase or in solution, these two synthesis techniques being well

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 known to those skilled in the art. These techniques are described in particular by Atherton and Shepard in Solid Phase Peptide Synthesis (IRL Press Oxford, 1989) and by Houbenweyl in method der organischen chemie edited by E. Wunsch vol, 15-1 and II thieme, Stuttgart, 1974, as well as in the following articles, the entirety of which is incorporated herein by reference: Dawson PE et al (Synthesis of Proteins by Native Chemical Ligation Science 1994; 266 (5186): 776-9); Kochendoerfer GG et al (Chemical Protein Synthesis, Curr Opin Chem Biol 1999; 3 (6): 665-71); and Dawson PE et al Synthesis of Native Chemicals by Chemical Ligation, Annu Rev Biochem 2000; 69: 923-60.

 The polypeptide according to the invention can also be produced by genetic engineering techniques well known to those skilled in the art. When the polypeptide according to the invention is produced by genetic engineering, it comprises at the NH 2 -terminal end of the sequence of formula I an additional methionine residue corresponding to the translation of the first initiation codon. These techniques are described in detail in Molecular Cloning: a molecular manual by Maniatis et al, Cold Spring Harbor, 1989).

Conventionally, the DNA sequence coding for the polypeptide according to the invention can be produced by the PCR technique in which the N and C sequences are first amplified independently of one another and then are in a second step paired and amplified again. The DNA sequence thus obtained is then inserted into an expression vector. The expression vector containing the sequence of interest is then used to transform a host cell allowing expression of the sequence of interest. The polypeptide produced is then isolated from the culture medium by standard techniques well known to those skilled in the art, such as precipitation with ethanol or ammonium sulfate, extraction with acid, chromatography of exchange of anion / cation, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Preferably, high performance liquid chromatography (HPLC) is used in the purification.

Depending on the expression system used (protein secreted or not) and according to the purification method, the purified polypeptide can be in various forms. It may be in a denatured or undenatured, monomeric or multimeric form. When it is in a denatured form it is possible to return its conformation open according to the invention using the method described in the examples given below. To obtain multimeric forms and in particular trimers, the purified polypeptide molecules must be placed in a medium which allows the molecules to be perfectly

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 soluble and essentially without interaction between them and preferably without secondary structure. For this purpose, it is possible to use detergents such as sodium dodecyl sulphate, lauryl sarcosine, guanidinium chloride, urea, sodium thiocyanate or chaotropic agents. The desired conditions can be promoted by the use of organic solvents or the use of acids. This first condition being satisfied, the sample is placed in a dialysis cassette to remove some of the detergents or chaotropic agents used, so as to promote interactions between the polypeptide monomers by keeping the molecules sufficiently solubility. In a second step, the formation of trimers being favored, the sample is completely dialyzed in a physiological medium which maintains the polypeptide in solution or suspension.

Trimeres of the polypeptide according to the invention are then obtained. Such a technique is described in detail in application WO00 / 08167.

 To carry out the synthesis of the polypeptide, any expression vector conventionally used for the expression of a recombinant protein may be used in the context of the present invention. This term thus encompasses both so-called living expression vectors such as viruses and bacteria as plasmid expression vectors.

 Vectors in which the DNA sequence of the polypeptide according to the invention is under the dependence of a strong, inducible or non-inducible promoter are preferably used. As an example of a promoter that may be used, mention may be made of the promoter of the T7 RNA polymerase.

 The expression vectors preferably include at least one selection marker. Such labels include, for example, the dihydrofolate reductase gene or the neomycin resistance gene for eukaryotic cell culture and the kanamycin, tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria.

 Exemplary expression vectors which can be used in the context of the present invention are the plasmids pET28 (Novagen) or pBAD (Invitrogen) for example; viral vectors such as: baculovirus, poxvirus, in particular the poxviruses described in patents US 5,942, 235, US 5,756, 103 and US 5,990,091, the entirety of which is incorporated herein by reference, the recombinant vaccinia viruses, in particular the recombinant viruses described in patents EP 83286, US 5,494, 807 and US 5,762,938 in which the DNA sequence coding for a polypeptide according to the invention is cloned.

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 To promote the expression and purification of the polypeptide, the latter may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to enhance stability and maintenance in the host cell.

 For the expression of the polypeptide, any host cell conventionally used in combination with the expression vectors described above may be used.

 Mention may be made, by way of non-limiting example, of E. coli, BL21 (IDE3), HB101, Top10, CAG 1139, Bacillus cells, eukaryotic cells such as CHO or Vero.

 Preferably, in the context of the present invention, the following expression vector / cell system will be used: pET (Cer) / BL21LamdaDE3, or BL21lamdaDE3 (RIL).

 Depending on the host cell used for expression of the polypeptide, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, the polypeptides according to the invention may also include an additional N-terminal methionine residue.

Once purified, the polypeptide according to the invention may advantageously be mixed with 2,2,2-trifluoroethanol (TFE). To do this, the polypeptide is preferably placed in an acidic buffer such as sodium formate at a pH = 2.5. The mixture thus formed contains from 10 to 50% by vol of TFE, preferably from 15 to 30% by weight. TFE flight. The TFE has the effect of increasing the rate of helicity of the polypeptide to bring it to a value close to or equal to 100%.

In addition to TFE, other organic solvents or detergents can also be used.

By way of example, mention may be made of: isopropanol or lyso phospholipid. Suitable amounts of these compounds can be readily determined by those skilled in the art.

Since these compounds are generally toxic and consequently not manageable, it is necessary to eliminate them before administration. The Applicant has demonstrated that these solvents or detergents could be easily removed by adding a carrier adsorbing the polypeptide. The carrier used in the context of the present invention corresponds to a pharmaceutically acceptable carrier which can be administered in humans. Examples of carriers that may be used in the context of the present invention include aluminum hydroxide, phosphate, or hydroxy-phosphate gels which are conventionally used as adjuvants in vaccines. The support

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 is used in large excess relative to the polypeptide to obtain a total adsorption of the latter.

Any other pharmaceutically acceptable carrier capable of adsorbing the polypeptide according to the invention could be used in the context of the present invention.

 Typically the required amount of pharmaceutically acceptable carrier is added to the polypeptide / TFE mixture and then the whole is incubated at room temperature for the time necessary to allow total absorption of the polypeptide on the support. The incubation time may vary from 15 minutes. at 3 o'clock. the mixture is then centrifuged 1 or 2 times at about 10000 g and the pellet is taken up in a solution that can be administered in humans, such as, for example, in the case of an injectable composition of PBS buffer or a physiological saline solution.

 The subject of the present invention is also the conjugates comprising a polypeptide according to the invention and a carrier protein or a carrier peptide.

 The carrier protein (or peptide) enhances the immunogenicity of the polypeptide according to the invention, in particular by increasing the production of specific antibodies. Said carrier protein (or peptide) preferably comprises one or more helper epitope (s).

By T helper epitope is meant a sequence of amino acids which, in the context of one or more molecules of the MHC class II, activates helper T cells. According to an advantageous embodiment, the carrier protein (or peptide) used improves the solubility in water of the polypeptide according to the invention.

 As the carrier protein, for example, phage surface proteins such as phage M13 protein pIII or pVIII may be used, bacterial surface proteins such as LamB, OmpC, ompA, ompF and PhoE proteins of E. coli. coli, B. Subtilis CotC or CotD protein, bacterial porins such as Neisseria gonorrheae porant P 1, PI porin or P2 of H. influenzae B, N. meningitidis B class 1 porin, K. pneumoniae P. pneumonia, lipoproteins such as B. bugdorfi OspA, S. pneumoniae PspA, N. meningitidis B TBP2, E. coli as well as the adhesin A of S. pneumoniae; heat shock proteins such as Hsp65 or Hsp71 from M. tuberculosis or Bovis or Hin 47 from H. Infuenzae type B. Also particularly suitable in the context of the present invention are detoxified bacterial toxins such as tetanus toxoid or diphtheria, subunit B cholera toxin, subunit B cholera toxin, subunit B P. aeruginosa endotoxin A or S. aureus exotoxin A.

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 As a carrier peptide, for example, the p24E, p24N, p24H and p24M peptides described in WO94 / 29339 and the PADRE peptides as described by Del guercio et al (Vaccine (1997) vo115 / 4 can be used for example in the context of the present invention. , p441-448).

 The carrier protein (or peptide) is linked to the N or C terminus of the polypeptide according to the invention by any conjugation method well known to those skilled in the art. In addition, the sequence coding for the carrier protein (or peptide) may advantageously be fused to the coding sequence for the polypeptide according to the invention and the resulting sequence may be expressed in the form of a fusion protein by any conventional method. All the genetic engineering techniques useful for this purpose are described in Maniatis et al. Said conjugates can be isolated by any conventional purification method well known to those skilled in the art.

 The subject of the present invention is also the DNA sequences coding for the polypeptides and the conjugates according to the invention as well as the expression vectors comprising said sequences and the host cells transformed by said vectors. The DNA sequences coding for the polypeptides according to the invention can be easily produced by PCR using as template the nucleotide sequence of a gp41 protein.

 Rather than extracting and purifying the polypeptide or conjugate expressed by the expression vector, it is often easier and sometimes more advantageous to use the expression vector itself in the vaccine according to the invention. The present invention therefore also relates to any expression vector as defined above. In such a case, the expression vector is devoid of a marker and preferably corresponds to a viral vector, in particular a poxvirus such as ALVAC or NYVAC.

 Any host cell as defined above transformed with such an expression vector is also included within the scope of the present invention.

 The subject of the present invention is also the antibodies directed against the polypeptides and conjugates as described above. The preparation of such antibodies is carried out by conventional techniques for obtaining polyclonal and monoclonal antibodies well known to those skilled in the art.

 These antibodies are particularly suitable for use in a passive immunization scheme.

 The present invention also relates to pharmaceutical compositions useful for the purpose of therapeutic and prophylactic immunization of infections related to

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 HIV. The compositions according to the present invention comprise at least one polypeptide, at least one conjugate or at least one expression vector as defined above, a pharmaceutically acceptable excipient or diluent and optionally an adjuvant.

 According to a preferred embodiment, the composition according to the invention further comprises a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier capable of adsorbing the polypeptide according to the invention may be used. A description of such media has been provided above. In the case where the pharmaceutically acceptable carrier is an aluminum salt, the latter provides both the support function and the adjuvant function.

 The amount of polypeptide, conjugate or vector in the composition according to the present invention depends on many parameters as will be understood by those skilled in the art, such as the nature of the carrier protein, the vector used or the route of the invention. administration. An appropriate amount is an amount such that a humoral immune response capable of neutralizing primary isolates of HIV is induced after administration of the latter. The amount of polypeptide to be administered is of the order of 10 μg to 1 mg, the selected amount varying depending on the route of administration. The amount of conjugate to be administered will be deduced from the amounts indicated above taking into account the PM of the carrier protein. The amount of expression vector to be administered is of the order of 10 to 5000 micrograms in the case of a non-viral vector and of the order of 10E4 to 10Eg TCID50 in the case of a viral vector.

 The pharmaceutical compositions according to the present invention may also contain an adjuvant. Any adjuvant or mixture of pharmaceutically acceptable adjuvants conventionally used in the field of vaccines can be used for this purpose.

By way of example, mention may be made of aluminum salts such as aluminum hydroxide or aluminum phosphate. Conventional auxiliaries such as wetting agents, fillers, emulsifiers, buffers, etc. can also be added to the composition according to the invention.

 The compositions according to the present invention may be prepared by any conventional method known to those skilled in the art. Conventionally, the antigens according to the invention are mixed with a pharmaceutically acceptable excipient or diluent, such as water or phosphate buffered saline. The excipient or diluent will be selected according to the chosen dosage form, mode and route of administration as well as pharmaceutical practice. Suitable excipients or diluents as well as pharmaceutical formulation requirements are described

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 in details in Remington's Pharmaceutical Sciences, representing a reference work in this field.

 The compositions mentioned above may be administered by any conventional route, usually used in the field of vaccines, such as parenteral (intravenous, intramuscular, subcutaneous, etc.). In the context of the present invention, preference will be given to injectable compositions for intramuscular administration. Such administration can be performed advantageously at the level of the muscles of the thigh or the arm. The compositions according to the present invention can also advantageously be administered orally. Administration via the nasal, vaginal or rectal mucosa may also be recommended in the context of the present invention. The administration can be carried out by the administration of a single dose or repeated doses, for example in OJ, at 1 month, 3 months, 6 months and 12 months.

OJ injections should preferably be used at 1 month and 3 months with a reminder whose periodicity can be easily determined by the attending physician.

 The pharmaceutical composition according to the present invention may advantageously be administered according to a dosage scheme comprising the co-administration of an expression vector according to the invention and a polypeptide according to the invention or according to a prime-boost scheme in which the vector according to the invention is administered first and the polypeptide as a booster injection. In these two regimens, the expression vector according to the invention may be replaced by any expression vector expressing one or more HIV antigens or epitopes different from the polypeptide according to the invention, and in particular by a poxvirus, preferably ALVAC. or NYVAC. Examples of ALVAC and NYVAC vectors that may be used for this purpose are the vectors described in US Pat. Nos. 5,942, 235, 5,756, 103 and 5,990,091; EP 83286, US 5,494, 807 and US 5,762,938. Bacterial vectors such as lactobacillus or salmonella can also be advantageously used in the context of compositions that can be administered orally. The use of these bacterial vectors for immunization purposes is described in detail in International Journal of Food Microbiology 41 (1998) 155-167 of PH Pouwels et al and Cell Vol 91,765-775, Dec, 1997 to A. Darji and al which we can refer for more details.

 The present invention also aims to cover a polypeptide, a conjugate or a vector as defined above and the pharmaceutical composition containing these compounds for their use as a medicament, in particular for the prophylactic and therapeutic immunization of the human body against infections related to HIV

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According to a preferred aspect, the subject of the present invention is the use of a polypeptide according to the invention for the immunization of the human body. The present invention therefore preferably relates to a method of administering said polypeptide to induce a specific humoral response.

 By a specific humoral response is meant a response comprising the production of antibodies directed specifically against the polypeptide according to the invention. The production of specific antibodies can be easily determined by standard techniques well known to those skilled in the art such as ELISA, RIA, Western blot.

The Applicant has surprisingly demonstrated that the polypeptide according to the invention is capable after administration of inducing antibodies capable of neutralizing primary isolates of HIV. These antigens are therefore valuable candidates for the development of a vaccine that can be used for the protection and / or treatment of many, if not all, HIV-infected or at-risk individuals.
Without wishing to be bound by any theory, the Applicant believes that the open conformation of the polypeptide according to the invention makes accessible domains of gp41 which are only accessible during the phenomenon of fusion of the viral and cellular membranes in the intermediate conformation which is adopted. transiently. These domains made accessible would be the target of the antibodies induced by the polypeptide according to the invention, the latter thus blocking the phenomenon of membrane fusion at a pre-fusogenic stage. The open conformation adopted by the polypeptide according to the invention thus mimics the conformation adopted by the intermediate state.

 The invention also relates to a diagnostic method comprising contacting a polypeptide according to the invention with a biological sample and detecting the antibody / polypeptide complexes that have formed. HIV + subjects contain anti-gp41 serum antibodies. An immunological test (such as an ELISA test in which the polypeptide according to the invention is fixed on the test plate and then put in contact with the serum to be tested and the antibody / polypeptide complexes are then detected) would thus make it possible to diagnose the subjects infected.

The present invention will be described in more detail in the examples which follow with reference to the appended figures in which:
Figure 1 is a schematic representation of the conformational change phenomenon of gp41 that precedes the fusion of cell and viral membranes.

 Figure 2 shows a spectrum obtained by the DSC technique.

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 Figure 3 shows a spectrum obtained by the circular dichroism technique.

 Figure 4 gives the plasmid map of pET-Cer (SEQ ID N 35).

 The examples described below are given purely by way of illustration of the invention and can in no way be considered as limiting the scope of the invention.

d'une matrice correspondant à un plasmide contenant la séquence d'ADN codant pour la région core de la gp41 (SEQ ID NO 34) en utilisant les paires d'amorces 5'N/3'N et 5'C/3'C telle que définies ci-dessous dans le tableau 1. Pour chacune des séquences, la réaction de PCR est mise en oeuvre dans les conditions suivantes : On mélange dans un premier temps les amorces, la matrice, le mélange de nucléotides et la Taq polymérase et on porte le mélange à 94 C pendant 2 minutes. Le mélange résultant est ensuite soumis à 25 cycles de PCR selon le schéma suivant : 94 30s, 55 30s, et 68 30s. Les produits ainsi amplifiés sont ensuite purifiés sur des colonnes QIAQUICK dans les conditions recommandées par le fabricant. Example 1 Construction of the DNA Sequences Coding for the Polypeptides According to the Invention
The DNA sequences coding for the polypeptides corresponding to the sequences SEQ ID N4, 5.6, 7.8 and 9 were obtained by PCR according to the following method:
The N and C sequences are first amplified by PCR from

of a template corresponding to a plasmid containing the DNA sequence coding for the core region of gp41 (SEQ ID NO 34) using primer pairs 5'N / 3'N and 5'C / 3'C As defined below in Table 1. For each of the sequences, the PCR reaction is carried out under the following conditions: The primers, the template, the nucleotide mixture and the Taq polymerase are first mixed together. the mixture is heated at 94 ° C. for 2 minutes. The resulting mixture is then subjected to 25 PCR cycles according to the following scheme: 94 30s, 55 30s, and 68 30s. The products thus amplified are then purified on QIAQUICK columns under the conditions recommended by the manufacturer.

 The two products thus purified which comprise overlapping sequences are subjected mixed and subjected to a PCR amplification reaction using the 5'N and 3'C primers as defined below in Table 1. The PCR reaction is set according to the following scheme: the purified N and C sequences, the Taq polymerase and the nucleotide mixture are mixed and subjected to 10 cycles: 94 30s, 55 30s, and 68 30s. The 5'N and 3'C primers are then added and the resulting mixture is subjected to 20 PCR cycles according to the following scheme: 94 30s, 55 30s, and 68 30s.

 The DNA sequences thus amplified are then purified on QIAQUICK columns under the conditions recommended by the manufacturer.

<Desc / Clms Page number 16>

raster 1: Primers used in PCR amplification reaction

<Tb>
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP> N028
<tb>. <SEP>5'N<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GCC <SEP> AGA <SEP> CAA <SEP> TTA <SEP> TTG <SEP> TCT <SEP> GG
<tb> * <SEP>3'N<SEP> CTC <SEP> CAT <SEP> CCA <SEP> GGT <SEP> CAT <SEP> GTT <SEP> ATT <SEP> ATC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TCT <SEP> TTC <SEP> CAC
<tb>#<SEP>5'c<SEP> GTG <SEP> GAA <SEP> AGA <SEP> TAC <SEP> CTA <SEP> AAG <SEP> GAT <SEP> AAT <SEP> AAC <SEP> ATG <SEP> ACC <SEP> TGG <SEP> ATG <SEP> GAG
##### <SEP> TGA <SEP> CCC <SEP> CBT <SEP> CCC
<tb> TCC <SEP> TTT <SEP> ATC <SEP> TAA <SEP> TTC <SEP> CAA <SEP> TAA <SEP> TTC
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP> N <SEP> 29
<tb>#<SEP>5'N<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GCC <SEP> AGA <SEP> CAA <SEP> TTA <SEP> TTG <SEP> TCT <SEP> GG
<tb>. <SEP>3'N<SEP> GTT <SEP> AAT <SEP> TTC <SEP> TCT <SEP> GTC <SEP> CCA <SEP> CTC <SEP> CAT <SEP> CCA <SEP> CTG <SEP> TTG <SEP> ATC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TC
<tb>. <SEP>5'C<SEP> GAT <SEP> ACC <SEP> TAA <SEP> AGG <SEP> ATC <SEP> AAC <SEP> AGT <SEP> GGA <SEP> TGG <SEP> AGT <SEP> GGG <SEP> ACA <SEP> GAG <SEP> AAA <SEP> TTA <SEP> AC
<tb>. <SEP>3'C<SEP> CCG <SEP> CTC <SEP> GAG <SEP> CTA <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> TGA <SEP> CCC <SEP> CBT <SEP> CCC <SEP> CBT <SEP> TT <SEP> ATC
<tb> TAA <SEP> TTC <SEP> CAA <SEP> TAA <SEP> TTC
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP> N030
<tb>. <SEP>5'N<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GCC <SEP> AGA <SEP> CAA <SEP> TTA <SEP> TTG <SEP> TCT <SEP> GG
<tb>. <SEP>3'N<SEP> CAT <SEP> CCA <SEP> GGT <SEP> CAT <SEP> GTT <SEP> ATT <SEP> ATC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TCT <SEP> TTC
<tb>. <SEP>5'C<SEP> GAA <SEP> AGA <SEP> TAC <SEP> CTA <SEP> AAG <SEP> GAT <SEP> AAT <SEP> AAC <SEP> ATG <SEP> ACC <SEP> TGG <SEP> ATG
<tb>. <SEP>3'C<SEP> CCG <SEP> CTC <SEP> GAG <SEP> CTA <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> TGA <SEP> CCC <SEP> CBT <SEP> CCC <SEP> CBT <SEP> TT <SEP> ATC
<tb> TAA <SEP> TTC <SEP> CAA <SEP> TAA <SEP> TTC
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP>? <SEP> 31
<tb>#<SEP>5'N<SEP> TTA <SEP> TTG <SEP> GAA <SEP> TTA <SEP> GAT <SEP> AAA <SEP> GCC <SEP> AGA <SEP> CAA <SEP> TTA <SEP> TTG <SEP> TCT
##### <SEP> TGA <SEP> CCC <SEP> CBT <SEP> CCC
<tb> TCC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TCT <SEP> TTC <SEP> CAC
<tb>. <SEP>5'C<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GGA <SEP> TGG <SEP> ATG <SEP> GAG <SEP> TGG <SEP> GAC <SEP> AGA <SEP> G
<tb>. <SEP>3'C<SEP> AGA <SEP> CAA <SEP> TAA <SEP> TTG <SEP> TCT <SEP> GGC <SEP> TTT <SEP> ATC <SEP> TAA <SEP> TTC <SEP> CAA <SEP> TAA
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP> N032
##### <SEP> ATT <SEP> GTC <SEP> TGG
##### <SEP> TGA <SEP> CCC <SEP> CBT
<tb> CCC <SEP> TCC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TCT <SEP> TTC <SEP> CAC
<tb>. <SEP>5'C<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GGA <SEP> TGGATG <SEP> GAG <SEP> TGG <SEP> GAC <SEP> AGA <SEP> G
<Tb>

<Desc / Clms Page number 17>

<Tb>
<tb>. <SEP>3'CCCA<SEP> GAC <SEP> AAT <SEP> AAT <SEP> TGT <SEP> CTG <SEP> GCT <SEP> GCC <SEP> CAT <SEP> TTA <SEP> TCT <SEP> AAT <SEP> CBT
<tb> Construction <SEP> of <SEP> SEQ <SEP> ID <SEP> N033
<tb>. <SEP>5'N<SEP> GGG <SEP> CAA <SEP> GTT <SEP> TGT <SEP> GGA <SEP> ATT <SEP> GGG <SEP> CCA <SEP> GAC <SEP> AAT <SEP> TAT <SEP> TGT <SEP> GTG <SEP> GCCG <SEP> CTC <SEP> GAG
<tb>. <SEP>3'N<SEP> CTA <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> ATG <SEP> GTG <SEP> TGA <SEP> CCC <SEP> TBT <SEP> CCCTCC <SEP> CTT <SEP> TAG <SEP> GTA <SEP> TCT <SEP> TTC
<tb> CAC
<tb>. <SEP>5'C<SEP> CAT <SEP> GCC <SEP> ATG <SEP> GGA <SEP> TGG <SEP> ATG <SEP> GAG <SEP> TGG <SEP> GAC <SEP> AGA <SEP> GCCA <SEP> GAC <SEP> AAT <SEP> AAT <SEP> TGT <SEP> CTG
<tb>. <SEP>3'C<SEP> GCC <SEP> CAA <SEP> TTC <SEP> CAC <SEP> AAA <SEP> CTT <SEP> GCC <SEP> C
<Tb>

<Desc / Clms Page number 18>

Example 2 Cloning of the DNA Sequences of Example 1 into an Expression Vector

fragment PCR, puis clonées dans un vecteur pET-cer selon le mode opératoire suivant : le fragment PCR et le vecteur digéré par les enzymes Ncol et Xhol sont ligués entre eux puis utilisés pour transformer une souche bactérienne XLI par électroporation. L'obtention de la construction est vérifiée par minipréparation du plasmide et séquençage. The DNA sequences produced in Example 1 are digested with NcoI and XhoI under standard conditions using 10 units of each of the enzymes per 1 g of

PCR fragment, then cloned into a pET-cer vector according to the following procedure: the PCR fragment and the vector digested with the Ncol and XhoI enzymes are ligated together and then used to transform an XLI bacterial strain by electroporation. Obtaining the construct is verified by minipreparation of the plasmid and sequencing.

permettent d'obtenir ce fragment encadré par les sites AscI et PacI. The pET-cer vector used is constructed from the Novagen pET28 vector. The pET28c commercial vector was amplified by PCR using 2 primers located on either side of the region corresponding to the origin f 1, so that the amplified product corresponds to almost all the original vector minus the region comprising the origin f1. The unique AscI and PacI restriction sites are provided respectively by the 2 primers used for the amplification. In parallel, the cer fragment is amplified using 2 primers which

allow to obtain this fragment framed by the sites AscI and PacI.

Vector and fragment cer are digested by the enzymes AscI and PacI then ligated together. This vector comprises in particular an expression cassette under the control of the T7 promoter, a polylinker downstream of the T7 promoter for cloning the gene of interest, the fragment cer located downstream of the polylinker, to reduce the multimerization of the plasmids, a term T7 transcription terminator and kanamycin resistance gene.

Positive upregulation of the promoter is achieved in the presence of T7 RNA polymerase.

The plasmid map of the plasmid pET-cer is given in FIG. 4 (SEQ ID NO. 35).

Example 3: Preparation of the SEO ID polypeptide 7 according to the invention
EXPRESSION Expression of the Plasmid Derived from Example 2 Containing the Sequence Encoding the Polypeptide SEQ ID? 7 is carried out in a modified strain of E. coli ie: BL21 RILXDE3.

This strain is enriched in rare tRNAs (ARG, ILE, LEU), it contains the gene encoding the T7 RNA polymerase which is under the control of the inducible lac UV5 promoter by addition of IPTG at a concentration of 1 mM. strain is transformed by the plasmid according to the protocol comprising the following steps: transplanting 3 colonies into 10 ml of LB supplemented with

<Desc / Clms Page number 19>

kanamycine àune concentration de 25g/JlI ; Incubation une nuit à 37 C ; réensemencer la préculture au 1 : 100 dans 15ml de LB additionné de kanamycine à une concentration de 2511gel ; laisser pousser jusqu'à D0600 de 0,5 ; Prélever 1 ml pour vérifier la D0600 ; prélever 7 ml pour l'échantillon non induit ; induire les 7 autres ml avec 1 mM d'IPTG et induction 3H à 37 C.

kanamycin at a concentration of 25 g / ml; Incubation overnight at 37 C; reseed the preculture to 1: 100 in 15ml of LB supplemented with kanamycin at a concentration of 2511gel; allow to grow to D0600 of 0.5; Take 1 ml to check the OD600; take 7 ml for the uninduced sample; induce the other 7 ml with 1 mM IPTG and induction 3H at 37 ° C.

The same protocol was implemented on several liters of culture to produce a large quantity of bacteria for purifying the polypeptide according to the invention.

 2-Purification the cell pellet, formed of the bacteria harvested in 500 ml of culture medium, is thawed and taken up in 100 ml of 50 mM Tris-HCl buffer at pH 8.0 in the presence of a protease inhibitor (Péfabloc, Interchim) at the concentration 100uM. Lysozyme is added at a concentration of 100 μg / ml and the mixture is incubated for 30 minutes at room temperature with stirring. The cells are then broken by sonication (4 cycles of two minutes) with an approximate power of 150 Watts. Benzonase (DNase, Merck) is then added with 1 mM MgCl2 and the mixture is incubated for 20 minutes at room temperature with stirring. After centrifugation (20 min at 10,000 g), the polypeptide according to the invention is in the insoluble fraction in the form of inclusion bodies. These are washed with 50mM Tris-HCl buffer at pH 8.0 and centrifuged for 15min at 10000g.

The polypeptide according to the invention can then be purified by one of the following two methods: Method 1: After removal of the supernatant, the solubilization of the centrifugation pellet consisting essentially of the inclusion bodies is carried out in one hour at room temperature by stirring soft in the presence of 50ml of CAPS buffer at pH 11.0 containing 1% N-lauryl sarcosine.

filtre de porosité 0, 45um puis chargée sur une colonne Hi-Trap Iml (Pharmacia). Ces supports de chromatographie d'affinité chélatent des atomes de Nickel sur lesquels se fixent les résidus histidine de l'extrémité C-terminal du polypeptide. Après lavage, l'élution du polypeptide est obtenue avec du tampon acide formique 50mM à pH 2,5. The solubilized fraction is then dialyzed at 4 ° C. against 50 mM Tris-HCl buffer at pH 8.0 containing decreasing concentrations of detergent (0.2% final) and filtered through a filter.

filter of porosity 0, 45um then loaded on a Hi-Trap Iml column (Pharmacia). These affinity chromatography supports chelate nickel atoms on which the histidine residues of the C-terminus of the polypeptide are fixed. After washing, the elution of the polypeptide is obtained with 50 mM formic acid buffer at pH 2.5.

<Desc / Clms Page number 20>

Process2: After removal of the supernatant, the solubilization of the centrifugation pellet consisting essentially of the inclusion bodies is carried out for one hour at room temperature by gentle stirring in the presence of 30 ml of 50 mM Tris-HCl buffer pH 8.0, 8M urea. After filtration on a 0.45 μm porosity filter, the fraction is loaded onto a 1ml Hi-Trap column (Pharmacia). After washing, the elution is carried out with Tris-HCl buffer 50mM pH8, 0 + Urea 8M + imidazole 500mM. The eluted fraction is then dialyzed against 50 mM formic acid buffer at pH 2.5.

EXAMPLE 4 Preparation of SEO ID N6 Polypeptide According to the Invention The polypeptide is expressed using the protocol described in Example 3 and then purified according to the procedure described below.

The cell pellet, formed of the bacteria collected in 500 ml of culture medium, is thawed and taken up in 100 ml of 50 mM Tris-HCl buffer at pH 8.0 in the presence of a protease inhibitor (Péfabloc, Interchim) at a concentration of 100 μM. Lysozyme is added at the concentration of 100 μg / ml and the mixture is incubated for 30 minutes at room temperature with stirring. The cells are then broken by sonication (4 cycles of two minutes) with an approximate power of 150 Watts. Benzonase (DNase, Merck) is then added with 1 mM MgCl2 and the mixture is incubated for 20 minutes at room temperature with stirring. After centrifugation (20 min at 10,000 g), the polypeptide according to the invention is predominantly in the soluble fraction. After filtration on a 0.45 μm porosity filter, the fraction is loaded onto a 1ml Hi-Trap column (Pharmacia). After washing, the elution is carried out with 50 mM Tris-HCl buffer pH 8.0 + 500 mM imidazole. The eluted fraction is then dialyzed against 50 mM Tris-HCl buffer buffer pH 8.0.

The polypeptide at a concentration of 0.4 mg / ml is then placed in a 50 mM sodium formate buffer at pH 2.5. A TFE / polypeptide mixture is prepared by adding 1 vol. TFE for 1 vol. of polypeptide solution. The mixture thus obtained is incubated at room temperature for 15 minutes. 6 mg of Al phosphate are added together with 50 mM sodium formate buffer to obtain a final volume of 0.5 ml.

The mixture is then incubated at 4 ° C. with stirring for a time which can vary between 15 min. and 3 hours to allow total adsorption of the protein on the aluminum phosphate. After the adsorption step, the mixture is centrifuged for 5 minutes at about 10000g. The supernatant is removed and the pellet is resuspended in the original volume by adding the necessary amount of PBS buffer. A second centrifugation and a

<Desc / Clms Page number 21>

 recovery in the same buffer eliminates the last traces of formic acid and TFE.

The preparation thus obtained forms the administrable pharmaceutical composition.

EXAMPLE 5 Analysis by DSC of the Polypeptides According to the Invention The analyzes were carried out on a VP microcalorimeter (Microcal, Northampton, Massachusetts, USA) as follows: Preparation of the samples: Polypeptide concentration: 0.4-0.7 mg / ml, determined by the MicroBCA method (Pierce, Rockford, Illinois, USA). The polypeptide is filtered before analysis (cutoff threshold of 0.22 gm), degassed for 8 min. in the Thermovac (Microcal, Northampton, Massachusetts, USA) and then thermostated at 24 C. The samples are in 50mM Na formate buffer pH2.5.

Recording: After warming up the device which consists of 6-10 thermocycles of the buffer against buffer, the polypeptide is placed in the measuring cell when the temperature of the cell is 25 C.

The parameters of the thermocycles are as follows: (1) 15 min. balancing at 5 C; (2) increasing the temperature of 5 C-130 C with a speed of 85 C / H; (3) balancing at 130 ° C. and (4) cooling at 130 ° C. at 5 ° C. (maximum speed) comprising the step of loading the next sample at a temperature of 25 ° C. for the next measurement.

 The analysis was carried out on the ectodomain of gp41 LAI consisting of amino acids AA25-AA157 and on the polypeptides SEQ ID N031 and SEQ ID No. 30 according to the invention.

The protein concentration was 0.51 mg / ml for the whole ectodomain of gp41 and 0.63 mg / ml for the polypeptides according to the invention.

<Desc / Clms Page number 22>

The results shown in Figure 2 are given in Table 2 below: Table 2:

<Tb>
<tb> Sample <SEP> T) <SEP> AH <SEP> AHVH
<tb> [difference <SEP> type <SEP> 0, <SEP> 2 C] <SEP> (Kcal / mol) <SEP> (Kcal / mol)
<tb> Ectodomain <SEP> integer <SEP> of <SEP> gp41
<tb> 110, <SEP> 4 <SEP> 61 <SEP> 228
<tb> Polypeptide <SEP> SEQ <SEP> ID <SEP> N031
<tb> 101, <SEP> 9 <SEP> 58 <SEP> 140
<tb> Polypeptide <SEP> SEQ <SEP> ID <SEP> N030
<tb> 90, <SEP> 3 <SEP> 59 <SEP> 152
<Tb>
Example 6 Circular Dichroism Analysis of the Polypeptides According to the Invention
The analyzes were carried out on a spectropolarimeter (Jasco, Tokyo, Japan) under the following conditions: temp: 25 C; Vessel: optical path: O, lmm; Concentration of polypeptide: 1-2 mg / ml. The polypeptide is placed in the vat and the spectrum is recorded at a scanning rate of 10 nm / min. The spectra obtained are analyzed by Spectra Analysis software from Jasco. The spectrum of the buffer is used to correct the background noise.

 The assay was performed on the entire ectodomain of LAI gp41 (e.g., AA25-157) and the SEQ ID? 31 according to the invention.

 The results shown in FIG. 3 show that the entire ectodomain has a helicity rate of 69%, the polypeptide according to the invention has a helicity rate of 70% and the polypeptide mixed with TFE has a helicity rate. 100%.

semaines d'intervalle (dans les cuisses) à raison de 0, 25ml dans le biceps femoris de chaque cuisse, avec 20ug des polypeptides SEQ ID Ni28, SEQ ID N32, SEQ ID ? 30 ou SEQ ID ? 31 selon l'invention, traités ou non par 30% de TFE, en présence de 6mg de phosphate d'aluminium. Les sérums des animaux sont prélevés avant immunisation après deux immunisations et en fin de test de façon à suivre la montée des anticorps. Example 7: Guinea pig immunization test Eight groups of five guinea pigs are immunized three times intramuscularly at three

weeks apart (in the thighs) at a rate of 0, 25 ml in the biceps femoris of each thigh, with 20 μg of the polypeptides SEQ ID Ni28, SEQ ID N32, SEQ ID? 30 or SEQ ID? 31 according to the invention, treated or not with 30% TFE, in the presence of 6 mg of aluminum phosphate. The sera of the animals are taken before immunization after two immunizations and at the end of the test in order to follow the rise of the antibodies.

The neutralizing power of the antibodies obtained is determined by the technique of C. Moog as described above.

Claims (15)

CLAIMS. A polypeptide comprising a sequence of formula 1: wherein N represents the amino acid sequence 30 to 77 of gp41, C represents the amino acid sequence 117 to 154 of gp41, SI and S2 are independently of each other either absent or represent a sequence

 of amino acids such that the sequence of formula 1 adopts an alpha helix conformation as determined by the SOPMA software under the following conditions: number of conformational states = 4 Similarity limit = 8 and, Width of the window = 70

 n = 0 or 1; m = 0 or 1 and m + n = 1 or 2. 2. A polypeptide according to claim 1 wherein SI is absent or represents the amino acid sequence D, DQ, DQQ, DQQL or DNNMT and S2 is absent or represents the amino acid sequence W, WA, WAS, WASL or WASLW. The polypeptide of claim 1 or 2 wherein m + n = 1. 4. A polypeptide according to any one of claims 1 to 3 wherein N is SEQ ID N26 and C is SEQ ID? 27. 5. A polypeptide according to any one of claims 1 to 4 comprising a further sequence of formula (G) aS- (H) b wherein G represents a glycine residue, H represents a histidine residue, a is greater than or equal to 4 and b is greater than or equal to 6, said sequence being amide bonded to the NH 2 or COOH terminus of the polypeptide. <Desc / Clms Page number 24>  6. Conjugate comprising a polypeptide according to any one of claims 1 to 5, conjugated to a protein or a carrier peptide. 7. A DNA sequence coding for a polypeptide according to any one of claims 1 to 5 or a conjugate according to claim 6.  An expression vector comprising the DNA sequence of claim 7. 9.-host cell containing the vector according to claim 8. 10. A process for the preparation of a polypeptide according to any one of claims 1 to 5 comprising the expression of said polypeptide from a host cell as defined in claim 9. 11. A pharmaceutical composition comprising at least one polypeptide according to one of claims 1 to 5, or at least one conjugate according to claim 6 or at least one expression vector according to claim 8, a pharmaceutically acceptable excipient and optionally an adjuvant. . 12. Pharmaceutical composition according to claim 11 orally administrable. 13. The pharmaceutical composition of claim 11 or 12 further comprising a pharmaceutically acceptable carrier. A polypeptide according to any one of claims 1 to 5 for use as a medicament. 15.-Polypeptide according to claim 14 for the immunization of the human body against HIV-related infections