FR2629459A1 - PF11 to PF19 peptides from an HIV retrovirus - process for synthesising these peptides - their use especially in diagnosis - Google Patents

Abstract

The invention relates to peptides capable of forming an immunological complex with antibodies to protein F from a retrovirus capable of causing LAS or AIDS, characterised in that they contain at most 70 amino acids and possess an amino acid sequence corresponding to the C-terminal part of the protein F from an HIV or to a variant of this sequence, however such that it is immunologically recognised by antibodies to protein F from HIV retrovirus.

Description

PEPTIDES PF11 TO PF19 OF AN HIV RETROVIRUS - METHOD OF
SYNTHESIS OF THESE PEPTIDES - THEIR USE IN PARTICULAR
FOR DIAGNOSIS
The invention relates to peptides having immunological properties, where appropriate immunogenic, in common with the F protein of a retrovirus capable of causing in humans lymphadenopathy syndromes (ALS) susceptible to be relayed by a syndrome of acquired immunodeficiency (AIDS).

 So far, several retroviruses with a responsibility for the development of ALS or AIDS have been isolated and characterized. Thus a first virus called LAV-1 or HIV-1 has been isolated and described in patent application GB.83 / 24,800 and an application EP.84 / 401,834 of 4 / O9 / 84. This virus has also been described by F. Barre Sinoussi et al. in Science, 220, 45-99, 20, pages 868-871.

Variants of this HIV-1 virus designated by
LAV ELI and LAV MAL, have also been isolated, characterized and described in the EP patent application. 84 / 401,834.

The isolation and characterization of retroviruses belonging to a distinct class and having only a reduced immunological relationship with the above, have been described in European Patent Application No. 87 / 400.151.4 published under No. 239,425. These retroviruses have been grouped under the designation
HIV-2, have been isolated from several African patients with symptoms of lymphadenopathy or
AIDS.

The study of these retroviruses HIV-1 and HIV-2 led to the obtaining of their complementary DNA sequences and then to the characterization of the specific nucleotide sequences coding for other proteins than those described previously, in particular for the protein F of HIV-1 described in the Bruno publication
Guy et al. Nature 1987, vol. 33, p. 266-269.

 The antigenic properties of the F protein with respect to antibodies contained in a serum of a patient infected with a retrovirus capable of causing ALS or AIDS and more particularly with respect to antibodies directed against this protein were known. F.

 The inventors have found that certain peptide sequences selected from the known sequence of the F protein, for different isolates of HIV-1 or HIV-2 retrovirus, are of particular interest for the detection of an infection by one of the retroviruses. described above.

In a very interesting way, the inventors have noticed that certain peptides of the protein
F (also referred to as PF) are recognized by antibodies induced in a host infected with an HIV-type retrovirus at a very early stage of infection with the virus.

 This observation demonstrates the need, if any, that such peptides may represent for the detection at an early stage of an infection in humans by a retrovirus of classes HIV-1 or HIV-2.

In this respect, peptides of the F protein of a retrovirus capable of causing a
ALS or AIDS, which have immunological properties, in common with those of this F protein and which have the capacity to be recognized by antibodies against this protein, present in a biological sample, in particular a tissue or a biological fluid, a host infected with an HIV retrovirus in particular of the class
HIV-1 or class HIV-2.

 The invention is further directed to peptides of the F protein of an HIV-1 or HIV-2 retrovirus, having immunogenic properties, or capable of being rendered immunogenic in vivo.

 The peptides of the invention can be obtained by isolation from the complete sequence of the F protein of a specific retrovirus or by chemical synthesis.

FIG. 1 represents the peptide sequence of the F protein for two isolates, one corresponding to the HIV-2 retrovirus and the other to the HIV-1 retrovirus. Matches between amino acids and their one-letter code are as follows
M Methionine
Leucine
I Isoleucine
V Valine
Phenylalanine
S Serine
P Proline
T Threonine
Alanine
Y Tyrosine
Histidine
Q Glutamine
N Asparagine
K Lysine
Aspartic Acid
E Glutamic acid
Cysteine
W Tryptophan
R Arginine
Glycine
The peptides of the invention, obtained optionally after modification of the amino acid sequence, from an isolate of a retrovirus or obtained by synthesis are such that they have a sequence or (homology) with the corresponding peptide sequence of the F protein of a retrovirus of class HIV-1 or / and a retrovirus of class HIV-2, sufficient for the immunological properties of this sequence vis-à-vis the antibodies formed in a biological medium (including a serum) from a host infected with one or other of the HIV-1 viruses or
HIV-2 are preserved.

 In this regard, the peptides according to the invention, capable of forming an immunological complex with anti-F protein antibodies of a retrovirus capable of causing ALS or AIDS, are characterized in that they contain at most 70 amino acids forming a sequence corresponding to a portion of the F protein of an HIV retrovirus or a variant of this sequence nevertheless as it. is immunologically recognized by anti-F protein antibodies of HIV retrovirus.

In another embodiment of the invention, the peptide is selected according to its ability to be recognized by anti-protein antibodies
F of a host specifically infected with either HIV-1 or HIV-2 retrovirus.

A first peptide PF16 (HIV-1) according to the invention is characterized by all or part of the formula
XGM-DE-VL-WFS-LA-H-ARE-HPE-K-CZ in which - dashes "-" - "are variable amino acids selected so that the final peptide retains its ability to to be recognized by antibodies formed during an infection with one of the previous retroviruses, the X groups represent either a free NH 2 or amide group, especially by one or two alc.oyl groups comprising from 1 to 5 carbon atoms, or a peptide group comprising from 1 to 5 amino acids, whose N-terminal amino acid itself has a free NH 2 or amide group as previously indicated, and the Z groups represent either a free -OH or alkoxyl group and containing then an alkyl group comprising from 1 to 5 carbon atoms, ie a peptide group comprising from 1 to 5 amino acids, whose amino acid Cterminal itself has a free -OH or alkoxyl group, as previously indicated, the groups of 1 to 5 amino acids, if any, contained in X or Z or in the s both at the same time as the amino acids re nplacement dashes being such that their presence is not incompatible with the immunological properties, where appropriate immunogenic, peptides formed, including their ability to be recognized by human sera containing antibodies against the F protein of an HIV-1 retrovirus and more particularly monospecific antibodies against the above peptide.

Preferred peptides corresponding to the above structure are as follows
XGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNCZ
XGMEDAEKEVLVWRFDSKLAFHHMARELHPEYYKDCZ
XGMEDAEREVLKWKFDSSLALRHRAREQHPEYYKDCZ
XGMEDP ERQVLKWRFNSRLAFEHKAREMHPEFYKN Z
Particularly preferred peptides are given by the following amino acid sequences
GMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC GMEDAEKEVLVWRFDSKLAFHHMARELHPEYYKDC
GMEDAEREVLKWKFDSSLALRHRAREQHPEYYKDC GMEDPERQVLKWRFNSRLAFEHKAREMHPEFYKN
Other peptides belonging to the F protein covered by the invention characterized by their ability to form an immunological complex with antibodies directed against the F protein contained in a human serum infected with an HIV-1 retrovirus are the following peptides: : XMGGXWSKSSVVGWPTVRERMRRAEPAADGVGAYCZ
PF12
XGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLEKHGAITSSNTAATNAA
CAWLEAQEEEEVGZ Pu 13: XASRDLEKHGAITSSNTAATNAACAWLEAQEEEEZ
PF14: XCVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILZ
PF15
XCGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVZ
PF17
XCYKLVPVEPDKVEFANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVAR
ELHPEYFKNCZ
PF18: XCKGGLEGLIHSQRRQDILDLWIVHTQGYFPDZ
The invention still enganges variants of these peptides, insofar as they retain their immunological properties with respect to the antibodies directed against the F protein, present in a human serum infected with an HIV-1 retrovirus. variants of these peptides are more particularly the peptides obtained by bringing the above peptides into alignment with those of the peptides which correspond to them for the other isolates of HIV-1 described in FIG.

This figure represents the alignments of the F protein sequences for 4 isolates of the
AIDS. The LAVBRU isolate is taken as reference and the only differences of ARV2, LAVMAL and LAVELI with
LAVBRU are noted.

 The dashes correspond to deletions introduced into the alignments.

 Other peptides in accordance with the invention are capable of being recognized by antibodies formed in a host infected with a retrovirus of the HIV-2 class and are characterized in that they exhibit immunological, if appropriate immunogenic, properties in common with the following sequence PF16 (HIV-1) XKFDDPHGETLVWEFDPLLAYSYEAFIRYPEEFGHKZ in which X and Z have similar meanings to the preceding provided however that their choice allows the conservation of the capacity of the above peptide to be recognized by human sera containing antibodies against the F protein of an HIV-2 retrovirus and more particularly monospecific antibodies directed against the above peptide.

 Another peptide according to the invention capable of being recognized by antibodies formed in a host infected with an HIV-2 retrovirus is characterized in that it has immunological properties, where appropriate immunogenic, in common with the following PF19 sequence XCRGGLEGMFYSERRHRILNIYLEKEEGIIADZ in which X and Z have similar meanings to the above, provided however that their choice allows the conservation of the capacity of the above peptide to be recognized by human sera containing antibodies against the F protein of an HIV-2 retrovirus and more particularly monospecific antibodies directed against the above peptide.

 Other modifications of the various amino acid sequences described for the composition of the peptides of the invention, possibly deletions at the level of certain amino acids, may be envisaged provided that the modified sequence formed remains capable of being recognized by antibodies. monospecific against one or other of the relevant peptides, present in a biological medium, including human serum following infection with a retrovirus likely to cause ALS or AIDS.

 In particular, it is possible to use only part of the peptides described above insofar as a smaller sized sequence formed retains immunological properties, possibly immunogenic, with respect to antibodies directed against the F protein of a retrovirus. HIV.

 In a particular embodiment of the invention, some of the peptides PF11 to PF18 above are used in mixtures capable of being recognized by antibodies present in a human serum infected with a retrovirus class HIV-1. .

 In another embodiment or will make a mixture of peptides PF10 and PF19, capable of being recognized by antibodies present in a human serum infected with an HIV-2 retrovirus.

 It is also possible to make mixtures of peptides recognized by antibodies directed against a retrovirus of the HIV-1 type on the one hand and peptides recognized by a retrovirus of the HIV-2 type on the other hand.

 The invention also relates to compositions comprising such peptides alone or in admixture with other proteins or peptides encoded by the genome of a retrovirus capable of causing ALS or AIDS and their use in an invitro detection method. in a biological sample, infection with a retrovirus of the type HIV-1 or HIV-2.

 The invention also relates to the synthesis of the interesting peptides of the F protein.

 A principle of synthesis is, for example, to bring together a solid phase in which the sequence of the peptide is assembled and a liquid phase containing solvents and reagents. At each stage the separation between the growing peptide and the reagents is by simple filtrations and washes.

Initially, the solid support that carries a reactive group reacts with the carboxyl of an amino acid introduced with its blocked α-NH 2 (protected for example by t-butyloxycarbonyl), to establish a covalent bond. After deprotection of the amino function (for example by washing with an acid such as trifluoroacetic acid), a second protected amino acid is introduced to form by coupling the first peptide bond. The second amino acid, which provides the second amino-acyl of the sequence, is coupled from the C-terminal amino-acyl residue to the deprotected amine function of the first amino acid.
C-terminal of the chain. Preferably, the carboxyl function of this second amino acid is activated, for example by dicyclohexylcarbodiimide. By a series of reactions, deprotections and couplings, the synthesis progresses from the residue C- towards the N-terminal residue. When the desired sequence has been assembled, the peptide is removed from the solid phase by a specific reaction of cleavage of the peptide bond. -resin initially established. The detailed protocol of this synthesis is described in Example 1.

The method for synthesizing a peptide according to the above is characterized in that it comprises the following steps
a / bringing into contact a solid support carrying a reactive group capable of reacting with a carboxyl function, with the first C-terminal amino acid of the chain to be synthesized, protected at the level of the amine function which must subsequently be involved in the coupling with another amino acid, to avoid self-condensation
b / deprotection of the amine function of the amino acid previously fixed
c / coupling of the deprotected amine function of the amino acid at the end of step b / with the carboxyl function of a second amino acid, protected at its amine function and which provides the second aminoacyl residue then coupled to the C-terminal aminoacyl residue of the peptide to be synthesized
d / deprotection of the amine function of the second amino acid added and repetition of the c / coupling and d / protection stages, successively with the amino acids respectively corresponding to the successive aminoacyl residues until they reach the N-terminal aminoacyl residue of said peptide
e / separation of the peptide formed, by treatment of the solid phase carrying the synthesized peptide, in particular by means of hydrofluoric acid, once obtained the N-terminal amino acid
f / recovery of the peptide sequence from the solution.

 In a preferred embodiment of the preceding method, the deprotection step b / is followed by a neutralization step intended to render the nucleophilic amine function.

 In another particular embodiment, it is possible to resort to a second coupling step carried out after washing and neutralization of the peptide obtained at the end of step c /, by means of a coupling agent, in order to allow the reaction of the amine functions which had not reacted during the first coupling.

 For certain amino acids such as triptophane or cysteine, the deprotection of the reactive functions will be obtained after the separation of the peptide, of the resin respectively by means of a base or with a treatment with mercuric acetate.

 It is understood that the process for synthesizing the peptides according to the invention is not limited to the process previously described but on the contrary covers all the variants insofar as the peptide sequence thus synthesized retains the desired immunological properties.

According to another embodiment 9e of the invention, use will be made of the homogeneous solution synthesis technique described by HOUBENWEYL in the work entitled "Method der Organischen Chemie" (Method of the
Organic Chemistry) edited by E. Wunsch, vol. 15-I and II,
THIEME, Stuttgart 1974.

 This method of synthesis consists in successively condensing successively the successive aminoacyls in the required order, or in condensing aminoacyls and previously formed fragments already containing several aminoacyls in the appropriate order, or several previously prepared fragments. , it being understood that care has to be taken to protect in advance all the reactive functions borne by these aminoacyls or fragments, with the exception of the amine functions of one and the carboxyls of the other or vice versa, which are normally required to intervene in the formation of peptide bonds, especially after activation of the carboxyl function, according to methods well known in the synthesis of peptides. As a variant, it may be possible to resort to coupling reactions involving conventional coupling reagents, such as carbodiimide, such as, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. When the aminoacyl used has an additional acidic function (especially in the case of glutamic acid), these functions will for example be protected by t-butyl ester groups.

The invention also relates to a method for the in vitro detection of an infection with a retrovirus capable of causing ALS or AIDS, characterized in that it comprises the following steps
a) bringing at least one peptide according to the invention into contact with a biological sample to be tested, capable of containing antibodies against the F protein of an HIV retrovirus above, under conditions allowing the formation of a immunological peptide-antibody complex,
b) the detection of an immunological complex formed.

 To carry out the detection of the immunological peptide-antibody complex and thus make the diagnosis of the infection, it will be possible, for example, to resort to a radio-immunoassay. It is also possible to use an immunoenzymology method such as the ELISA method.

 In a first embodiment of the test for detecting a retrovirus infection capable of causing ALS or AIDS, the peptide used to diagnose the presence of anti-F protein antibodies is specifically recognized by a HIV-1 retrovirus.

 In a second embodiment of this detection test, the peptide used is specifically recognized by an HIV-2 retrovirus.

 In another embodiment of this test, it is chosen to use a mixture of peptides recognizing, on the one hand, antibodies formed against the F protein of an HIV-1 retrovirus and peptides recognizing, on the other hand, antibodies formed. against the F protein of an HIV-2 retrovirus. It is thus possible to perform a non-specific diagnosis of an infection with one or the other of the HIV-1 or HIV-2 retroviruses.

 To perform the diagnosis, the peptide may advantageously be attached to an ELISA plate.

 The invention also relates to the use of the peptides of the F protein, for purifying monospecific antibodies against the F protein.

 To carry out such a purification, for example by affinity chromatography, it is possible to operate as follows: fixation of the peptides used for the purification on a support, - passage on this support of a solution containing the antibodies which one seeks to purify to bring them into contact with the fixed peptides under conditions allowing the formation of a peptide-antibody immunological complex; said solution may be a polyclonal serum or a supernatant or an ascite containing monoclonal antibodies, - removal of unreacted constituents, - recovery of monospecific antibodies directed against the F protein.

 The invention also relates to monospecific antibodies directed against the F protein, characterized in that they are recognized by one or more peptides previously described.

The invention also relates to a kit for the invitro diagnosis of a retrovirus infection capable of causing ALS or AIDS, characterized in that it comprises
a peptide according to the invention, or a mixture of these peptides
the reagents for the constitution of a medium, in particular a buffered solution, suitable for the formation of an immunological reaction between the peptide (s) and the antibodies possibly present in a biological sample
one or more optionally labeled reagents capable of reacting with said peptide (s) for the detection of the peptide-antibody complex formed
if necessary, a reference biological medium, such as a serum, similar to the biological medium from the person possibly infected with an HIV retrovirus and devoid of antibodies recognizing the aforesaid peptides.

 The examples which follow illustrate the synthesis of the peptides according to the invention and their use in a diagnostic test for the presence of anti-F protein antibodies contained in human sera known to have been infected with an HIV-1 virus.

 Other features also appear in the figures.

FIG. 1 shows the alignment of the amino acid sequences of the F protein for the HIV-2 HIV-2 ROD.P and HIV-1 LAV.P isolates.

FIG. 2 represents the relationships between the overall efficiency of amino acid incorporation and the purity of the products obtained by solid phase synthesis (from Wang, S.S., 1972).

FIG. 3 represents the F protein of 4 retrovirus HIV-1 isolates.

EXAMPLE 1
SYNTHESIS. PURIFICATION AND CHARACTERIZATION OF PEPTIDES
1 / Nature of the solid support
For carrying out the synthesis, a good solid support is chosen so as to meet the following conditions: - to be insoluble in the solvents and reactants of the liquid phase - to be inert with respect to the reagents of the synthesis - to be sufficiently stable physically to be shaken and undergo filtrations - ensure excellent diffusion of reagents so as not to slow down the kinetics of a factor too important compared to that of the reactions in solution.

 Two kinds of resins have been used for the purposes of synthesis - a resin obtained by copolymerization of styrene in the presence of 1% divinylbenzene, the reactive function of which is benzydrylamine (-O-CHO-NH2); with a substitution of 0.2 mmol NH 2 / g of resin - a macroporous polyacrylamide resin whose reactive function is the amino methyl group (-CH 2 -NH 2) at 0.5 or 0.7 mmol NH 2 / g).

 2 / Nature of amino acids a / Protection of amino groups.

The α-NH 2 function of the added amino acid was protected to avoid the self-condensation reaction of the amino acids. The protective group used for this synthesis is tert-butyloxycarbonyl (Boc) which is removed with trifluoroacetic acid (TFA) 30% in dichloromethane (DCM).

b / Protection of lateral functional groups.

In contrast to a temporary protection of the amino functions, the functions carried by the side chains must be kept protected throughout the synthesis and, consequently, the protections must be non-labile in TFA 308 medium. On the other hand, they will be deprotected, in general, by the reagent used to cut the peptide-resin bond, hydrofluoric acid. The different protective groups corresponding to the amino acids used are shown in Table 1.

<Tb>

<SEP> Function, protected <SEP> t <SEP> Grouping <SEP> protector
<tb><SEP> Cl
<tb> OH <SEP> of <SEP><SEP> tyrosine <SEP> 3 <SEP> 1,6-dichlorobenzyl
<tb><SEP> CH <SEP> 29
<tb> cI
<tb> OH <SEP> of <SEP><SEP> Threonine <SEP> I
<tb> OH <SEP> dc <SEP><SEP> serine <SEP> t
<tb><SEP> 3 <SEP> benzyl <SEP> --- c <SEP> K
<tb><SEP> 1
<tb><SEP> - <SEP> COOH <SEP> of <SEP> aspartate <SEP> I
<tb><SEP> I
<tb><SEP> - <SEP> COOH <SEP> of the <SEP> glutamate
<tb><SEP> 3 <SEP> CH,
<tb><SEP> t <SEP> I
<tb> SH <SEP> of <SEP><SEP> cysteine <SEP> t <SEP> t. <SEP> butylmercapto <SEP> -S- <SEP> C- <SEP> CH
<tb><SEP> CH,
<tb> imida: ole <SEP> of <SEP> histidine <SEP> tosyl <SEP> S <SEP> 4 <SEP> CH3
<tb><SEP> -NH <SEP> of <SEP> arginine <SEP> i
<tb><SEP> 3 <SEP> o
<tb><SEP> -NH2 <SEP> of <SEP> the <SEP> lysine <SEP> t <SEP> ben: yloxycarbonyl <SEP> O <SEP> - <SEP> It
<tb><SEP> O-CH1
<tb> Indole <SEP> of <SEP> Tryptophan <SEP> t
<tb><SEP> t <SEP> fo
<tb><SEP> t <SEP> r.yl <SEP> -CHO
<Tb>
Tabicau I: Tertiary Function Protection of Urinated Amino Acids
in our syntheses.

3 / exPerimental DisPositif
The assembly of the peptides was carried out automatically in a synthesizer (APPLIED BIOSYSTEMS (registered trademark) Peptides Synthesizer model 430A) according to the general method described by MERRIFIELD).

4 / Protocol for incorporating a residue
The experimental protocol for performing a synthesis cycle comprises the following steps: a / - Deprotection of the amine functions
It is obtained after two 2-minute pretreatments followed by a 30-minute treatment with 30
TFA / CH2Cl2. The conditions used are normally sufficient to completely eliminate the clumps
Boc protectors of functions a-NH2, b / - Lavaae
The deprotected products were washed 5 times 2 minutes with CH 2 Cl 2.

c / - Neutralization
Necessary for deionizing the amine u-NH 2 of the peptide on the resin and rendering it nucleophilic to participate in the coupling reaction, the neutralization is obtained by two 2 minute pretreatments followed by a 10 minute treatment with 5 of DIEA / CH 2 Cl 2. (the
DIEA being Di-Isopropyl-Ethyl-Amine).

d / - First coupling
The first couple is made in an excess (3 times) of Boc amino acid (aa) and dicyclohexylcarbodiimide (DCC) in CH2Cl2 (2 hours). In the coupling whose mechanism is shown below, a side reaction gives a non-reactive urea derivative which depends on the nature of the solvent.

This reaction is rather weak in dichloromethane and is counterbalanced by the use of an excess of reagents (DCC and Boc-aa).

<tb><SEP> R12 <SEP> ,, <SEP> N-CN
<tb> Boc ~ NH ~ CH ~ C <SEP>. <SEP> O ~ N =
<tb> Boc <SEP> * <SEP> Amino Acid <SEP> dicyclohexykarbodiimide
<tb><SEP> R12
<tb> Boc <SEP> INH, CH, C
<tb><SEP> 0N = C <SEP> NKM <SEP>
<tb><SEP> R2 <SEP>"o
<tb><SEP> 80c ~ NH ~ <SEP> CH ~ C <SEP> 0 <SEP> ~
<tb><SEP> N ~ <SEP> C ~ NHt)
<tb><SEP> drift <SEP> urea <SEP> no <SEP> reagent
<tb><SEP> Rîl
<tb><SEP> NH2CKCH
<tb><SEP> 011 <SEP> P
<tb><SEP> v <SEP> r <SEP> aminoacyl. <SEP> resin
<tb><SEP> R2 <SEP> R, <SEP> 1
<tb> Bo <SEP> cNH-CH, C <SEP> -NH, CH <SEP> I (
<tb><SEP> ON <SEP> Or <SEP> P
<tb><SEP> Product <SEP> of <SEP> Coupling
<tb><SEP> NH ~ <SEP> C ~ NH0
<tb><SEP> dicyclohexyl <SEP> urea
<Tb>

Mechanism of coupling by dicyclohexylcarbamide.

- Lavaae
The reaction product is washed 5 times, 2 minutes with CH 2 Cl 2.

f / - New neutralization
To carry out this new neutralization, 2 pretreatments of 2 minutes followed by a treatment of 10 minutes with 5 of DIEA / CH2Cl2 are carried out.

 This new neutralization allows the activation of amines that would have escaped the first basic treatment.

- Washing
The product is washed 5 times 2 minutes with
CH 2 Cl 2 and 1 time 2 minutes with dimethylformamide (DMF).

h / - 2nd coupling
This second coupling involves as reagent the benzotriazole ester of Boc amino acid 1.5 times in excess in DMF (1 hour).

 The benzotriazole ester of Boc (aa) is prepared extemporaneously as follows - an equimolar mixture of DCC and hydroxybenzotriazole (HOBt) in dimethylformamide (DMF) is prepared and incubated for 10 minutes at 0 ° C.

Then, the Boc amino acid is added and the reaction is continued at OvC for ten minutes. The benzotriazole ester of the Boc amino acid thus formed is added to the reaction vessel.

i / - Wash
The product was washed again 5 times for 2 minutes with CH 2 Cl 2.

5 / Analytical checks during synthesis:
Since the synthesis proceeds without purification of the intermediates, it is imperative to check that the coupling reactions are complete.

The diagram in Figure 2 shows the importance of having complete couplings for the synthesis of large sequences; indeed, if a peptide of 40 residues is synthesized with an overall yield (deprotection, neutralization and coupling) of 98%, a mixture containing 46% of the desired peptide and 54% of different smaller peptides is obtained. If the yield is 99% on average per step the whole peptide will be present at 67%.

If the yield is 99.5% the desired peptide will then represent 82% and only 18% smaller peptide mixtures.

To verify the good performance of the coupling r three coupling tests can be carried out - Ninhydrin test (Kaiser, et al., 1970): an aliquot of peptidyl resin (approximately
IOmg) which is washed well with ethanol in small glass tubes. After centrifugation and removal of the ethanol, a solution of ninhydrin is added and allowed to react for 5 minutes at 95vu.

The appearance of a blue color (positive test) means incomplete coupling. If the color of the resin does not change the coupling occurred at least 99%.

- Fluorescamine test (Felix and Jimenez, 1973): a little more sensitive than that to ninhydrin, it can detect less than 1% of free NH2. An aliquot of peptidyl resin (10 to 20 mg) is washed successively with DCM, ethanol and DIEA 55 in DCM and then the fluorescamine is added. The reaction takes place in a basic medium (DIEA 58) for 10 minutes. After washing and drying, we look at the resin under a lamp.

UV at a wavelength of 366nm. The appearance of fluorescence means the presence of free NH2.

- Chloranyl test (Christensen, 1979): after the coupling of any amino acid to a proline, the first two tests can not be used because of their low sensitivity in the detection of secondary amines. Chloranyl (2,3,5,6 1,4-tetrachlorobenzoquinone) is used. To an aliquot of peptidyl resin (5 to 10 mg) is added 200 μl of acetone and 50 μl of a saturated solution of chloranyl in toluene; the whole is stirred for 5 minutes. if the green color is an incomplete coupling, the result of the test is that, if it remains positive after a second or third coupling, the α-NH 2 functions which have not reacted are acetylated to prevent deletion peptide formation, or, if negative, a new deprotection-coupling cycle is started. On the other hand, the amount of amines present on the resin is titrated at regular intervals (every two or three incorporated residues). This amount must remain constant and equal to the amount of amino acid initially fixed on the solid phase.

We use the picric acid salt method (Gisin, 1972): 10 to 20 mg of dried peptidyl resin are accurately weighed into a syringe and treated in the following way - 5 DCM washes - 3 3 minute treatments with a solution of picric acid (0.01 M in DCM) - 5 washes with DCM to remove the excess of picric acid - 5 treatments of 5 minutes with the DIEA (5% in DCM) which move the picrate related to the resin. The filtrates are collected in a 100 ml volumetric flask.

washing the resin with ethanol and then adding the flask to 100 ml with methanol - measuring the optical density at 358 nm: knowing the value of the eM (molar extinction coefficient) 358 nm (13800) the concentration of free α-NH 2 which could react with picric acid to form the corresponding salt is calculated.

6 / acetylation of the peptide on the resin
If the coupling of an amino acid remains incomplete after several reaction attempts, or if the synthesis of the peptide is complete and it is desired to block the NH 2 end of the peptide by the acetyl group (acetylation of the end N-terminal is made in order to have a peptide that mimics in terms of the charge, that which exists in the native protein), the peptide on the resin is acetylated under the following conditions - 5% DIEA neutralization - DCM washes - DMF - acetylation washes with a mixture of 1 ml of acetic anhydride (10 mmol) and 1.7 ml of DIEA (10 mmol) in the
DMF.The reaction lasts 20 to 30 minutes and is considered complete when the coupling test is negative.

7 / CouPure of the PeDtide-resin bond
The breakdown of the peptide-solid phase bond is made by anhydrous hydrofluoric acid (HF) at OC (Lenard and Robinson 1967, Sakakibara et al., 1967).

It is accompanied, if we chose protectors labile in the HF, the deprotection of the lateral chains.

The presence of anisole (10% by volume) is necessary to trap the free radicals and thus avoid the alkylation of certain residues, such as tyrosine, by the protective groups released. By using a benzhydrylamine resin, the peptide in its C-u amidated form is obtained at the end of the reaction. After evaporation of the hydrofluoric acid, the anisole is extracted with ether and the peptide is solubilized with acetic acid. The yield of the cleavage reaction is calculated from the results of amino acid analyzes giving the amounts of peptide before (peptide attached to the resin) and after (free crude peptide) cleavage by hydrofluoric acid. However some side chain protecting groups of some amino acids, such as formyl tryptophan are removed only by additional treatment.

8 / Deprotection of tryptophan
In formyl tryptophan form, the aromatic amino acid is suitably protected during synthesis against the risks of oxidation of the indole group. This indole group can be regenerated by the action of a base (1 M hydroxylamine, pH 9) for one hour in an aqueous medium (Ohno et al., 1972).

Peptides that have been synthesized are characterized by physicochemical properties given in Table 3 below

<SEP> HIV-1 <SEP> Mass <SEP> Mass <SEP> Molar <SEP>#<SEP> Theoretical <SEP> to <SEP> Load <SEP> net <SEP> Homology <SEP> with.
<tb> Localization <SEP> in <SEP> the <SEP> Molar <SEP> with <SEP> grps. <SEP> of <SEP> 280 <SEP> nm <SEP> to <SEP> pll <SEP> ne <SEP> neutral <SEP> the <SEP> sequence <SEP> SE <SEP> SE <SEP><SEP> pro- <SEP> in <SEP> g. <SEP> protection <SEP> in <SEP> cm-1M-1 <SEP> respondent <SEP> of
<tb> tine <SEP> F <SEP> represented <SEP> in <SEP> g. <SEP> HIV-2 <SEP> in <SEP>%
<tb><SEP> figure <SEP> 2
<tb> PF11 <SEP> 1-31 <SEP> 3636 <SEP> 5546 <SEP> 12350 <SEP> +3 <SEP> 26
<tb> PF12 <SEP> 1-66 <SEP> 7015 <SEQ> 10333 <SEQ> 16650 <SEW> -1 <SEP> 23
<tb> PF13 <SEP> 32-64 <SEP> 3578 <SEP> 5281 <SEP> 5550 <SEP> -4 <SEP> 18
<tb> PF14 <SEP> 65-109 <SEQ> 5222 <SEQ> 7480 <SEQ> 1750 <SEQ> +4 <SEP> 58
<tb> PF15 <SEP> 118-167 <SEP> 5886 <SEP> 8610 <SEP> 16600 <SEP> for <SEP> form
<tb><SEP> redemption
<tb><SEP> -1 <SEP> 56
<tb> PF16 <SEP> 171-205 <SEQ> 4408 <SEP> 6225 <SEP> 7550 <SEW> 0 <SEP> 40
<tb> PF17 <SEP> 141-205 <SE> 7830 <SE> 11769 <SEP> 9095 <SEP> +1 <SEP> 38
<tb> PF18 <SEP> 93-122 <SEP> 3735 <SEP> 5564 <SEP> 8300 <SEP> +1 <SEP> 40
<tb> PF19
<tb> of <SEP> HIV-2 <SEP> with <SEP> HIV-1
<tb><SEP> 125-154 <SEP> 3714 <SEP> 5756 <SEQ> 2750 <SEP> 0 <SEP> 40%
<Tb>

Table 3
EXAMPLE 2
SEROLOGY STUDY WITH PEPTIDE PF 16 PROTEIN
F, OBTAINED BY COMPARATIVE SYNTHESIS
The peptide was previously synthesized by the method described above. This peptide covers the 35 amino acid sequence of the C-terminal portion of the amino acid sequence of the retrovirus HIV-1 F protein.

The PF16 peptide has the following amino acid sequence in which acetamide (acm) represents a highly protective group of SH functions of cysteines
GMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC (acm)
For the purposes of the test, the different peptides PF16, PF19, PF13, PF17 used are diluted to a concentration of 2 mg / ml.

 On a strip of BIORAD paper for transblot, we put different spots: 11, 2ul, Siji, 101.

 After drying, a conventional Western Blot test is performed using sera of patients infected with the HIV-1 virus, positive for the F protein, at dilutions ranging from 1/100 to 1/300.

 The peptides tested make it possible to arrive at the results summarized in Table 2 with three different anti-F ++ sera (++ signifying a more pronounced reaction).

 Furthermore, the inventors have found that a monoclonal anti-F protein antibody of an HIV-1 retrovirus produced in the mouse recognizes the PF16 peptide of the invention in an ELISA test.

This shows the immunodominant character of the peptides according to the invention.

<SEP> SERUMS <SEP> POSITIVE, <SEQ> ANTI-PROTEIN <SEP> F <SEP> FROM <SEP> HIV-1
<tb><SEP> CHA <SEP> 1/300 <SEP> P.6. <SEP> 1/200 <SEP> 1545 <SEP> 1/200 <SEP> LOM <SEP> 1/100
<tb> PEPTIDE <SEP> 10 l <SEP> 5 l <SEP> 2 l <SEP> 1 l <SEP> 10 l <SEP> 5 l <SEP> 2 l <SEP> 1 l <SEP> 10 l <MS> 5 l <SEP> 2 l <SEP> 1 l <SEP> 10 l <SEP> 5 l <SEP> 2 l <SEP> 1 l
<tb> PF <SEP> 16 <SEP> ++ <SEP> ++ <SEP> ++ <SEP> ++ <SEP> + <SEP> - <SEP> - <SEP> - <SEP> ++ <SEP> - <SEP> - <SEP> - <SEP> ++ <SEP> ++ <SEP> - <SEP>
PF <SEP> 19 <SEP> ++ <SEP> ++ <SEP> - <SEP> - <SEP> + <SEP> tr <SEP> - <SEP> - <SEP> + <SEP> + <SEP> - <SEP> - <SEP> tr <SEP> tr <SEP> - <SEP>
PF <SEP> 13 <SEP> + <SEP> + <SEP> tr <SEP> tr <SEP> - <SEP> - <SEP> - <SEP> - <SEP> tr <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
PF <SEP> 17 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> tr <SEP> - <SEP> - <SEP> - <SEP> + <SEP> + <SEP> + <SEP> +
<Tb>

 TABEAU 2

Claims (18)

 A peptide capable of forming an immunological complex with anti-F protein antibodies of a retrovirus capable of causing ALS or AIDS, characterized in that it comprises at most 70 amino acids forming an amino acid sequence corresponding to a portion of the F protein of an HIV or a variant of this sequence nevertheless as it is immunologically recognized by anti-F protein retrovirus HIV antibodies.  2. Peptide according to claim 1, characterized by all or part of the sequence of formula  XGM-DE-VL-WFS-LA-H-ARE-HPE-K-CZ in which X and Z are OH or NH2 groups or, insofar as the immunological properties of the peptide lacking these groups are not 1 to 5 amino acid residues, and each of the indents corresponds to an aminoacyl residue chosen from those which make it possible to preserve the above-defined peptide the ability to be recognized by sera. humans containing antibodies against the F protein of an HIV-1 retrovirus and more particularly monospecific antibodies directed against the above peptide.  3. A peptide according to claim 1 or claim 2, characterized in that it consists of all or part of a peptide selected from the following peptides GMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC GMEDAEKEVLVWRFDSKLAFHHMARELHPEYYKDC GMEDAEREVLKWKFDSSLALRHRAREQHPEYYKDC GMEDPERQVLKWRFNSRLAFEHKAREMHPEFYKN  4. Peptide according to claim 1, characterized in that it is selected from the following XMGGKWSRSSVVGWPTVRERMRRAEPAADGVGAYCZ XGGKWSKSSVVGWPTVRERMRRAEPAADGVGAASRDLEKHGAITSSNTAATNAAC AWLEAQEEEEVG XASRDLEKHGAITSSNTAATNAACAWLEAQEEEEZ XCVGFPVTPQVPLRPMTYKAAVDLSHFLXEKGGLEGLIHSQRRQDILZ XCGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVZ XCYKLVPVEPDXVEFANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVAR ELHPEYFKNCZ XCKGGLEGLIHSQRRQDILDLWIVHTQGYFPDZ in which X and Z are OH or NH2 groups or insofar as the immunological properties of the peptide lacking these groups are not substantially modified, groups comprising from 1 to 5 amino acid residues selected from such that they make it possible to preserve the above-defined peptide with the ability to be recognized by human sera containing antibodies against the F protein of an HIV-1 retrovirus and more particularly monocospecific antibodies directed against the above peptides .  5. Peptide according to claim 1, characterized in that it has immunological properties, in common with those of the following sequence XKFDDPHGETLVWEFDPLLAYSYEAFIRYPEEFGHKZ in which X and Z are OH or NH2 groups or, insofar as the immunological properties of the peptide lacking these groups are not substantially modified, groups comprising from 1 to 5 amino acid residues, chosen in such a way that they make it possible to preserve the above-defined peptide with the capacity to be recognized by human sera containing antibodies against the F protein of an HIV-2 retrovirus and more particularly monospecific antibodies directed against the above peptide. above.  6. Peptide according to claim 1, characterized in that it has immunological properties in common with those of the following sequence XCRGGLEGMFYSERRHKILNIYLEKEEGIIADZ in which X and Z are OH or NH2 groups or, insofar as the immunological properties of the peptide lacking these groups are not substantially modified, groups comprising from 1 to 5 amino acid residues, chosen in such a way that they make it possible to preserve the above-defined peptide with the capacity to be recognized by human sera containing antibodies against the F protein of an HIV-2 retrovirus and more particularly monospecific antibodies directed against the above peptide. above.  7. Peptide mixture characterized in that it comprises a mixture of a peptide according to claim 2 or claim 4 and a peptide according to claim 5 or claim 6.  8. Kit for the in vitro diagnosis of a retrovirus infection capable of causing ALS or AIDS characterized in that it comprises  a peptide according to any one of claims 1 to 5, or a mixture according to claim 7;  the reagents for the constitution of a medium, in particular a buffered solution, suitable for the formation of an immunological reaction between the peptide (s) and the antibodies possibly present in a biological sample  one or more optionally labeled reagents capable of reacting with said peptide (s) for the detection of the peptide-antibody complex formed  - Where appropriate, a biological medium preferably, such as a serum, similar to the biological medium from the person possibly infected with an HIV retrovirus and lacking antibodies recognizing the aforesaid peptides.  9. Monospecific antibodies directed against the F protein, characterized in that they are recognized immunologically by one or more peptides according to one of claims 1 to 6.  10. Process for synthesizing a peptide according to any one of claims 1 to 6, characterized in that it comprises by the following steps  a / bringing into contact a solid support carrying a reactive group capable of reacting with a carboxyl function, with the first C-terminal amino acid of the chain to be synthesized, protected at the level of the amine function which must subsequently be involved in the coupling with another amino acid, to avoid self-condensation  b / deprotection of the amino function of the amino acid previously fixed  c / coupling of the deprotected amine function of the amino acid at the end of step b / with the carboxyl function of a second amino acid, protected at its amine function and which provides the second aminoacyl residue then coupled to the C-terminal aminoacyl residue of the peptide to be synthesized  d / deprotection of the amine function of the second amino acid added and repetition of the c / coupling and d / protection stages, successively with the amino acids respectively corresponding to the successive aminoacyl residues until they reach the N-terminal aminoacyl residue of said peptide  e / separation of the peptide formed by treatment of the solid phase carrying the synthesized peptide, in particular for example by means of hydrofluoric acid, once obtained the N-terminal amino acid  f / recovery of the peptide sequence from the solution.  11. Synthesis process according to claim 10, characterized in that step b / of deprotection is followed by a neutralization step intended to render the nucleophilic amine function.  12. Synthesis process according to claim 10 or 11, characterized in that the coupling step involves a coupling agent capable of activating the carboxyl function, preferably dicyclohexylcarboodiimide.  13. Synthesis process according to any one of claims 10 to 12, characterized by a second coupling step performed after washing and neutralization of the peptide obtained after step c /, by means of a coupling agent r to allow the reaction of the amine functions which had not reacted during the first coupling.  14. Synthesis process according to any one of Claims 10 to 13, characterized in that the protection of the tertiary functions of the amino acids is carried out by 2,6-dichlorobenzyl for the function OH for tyrosine, benzyl for OH function of threonine or serine or for COOH function of aspartate or glutamate, t-butyl mercapto for SH function of cysteine, tosyl for imidazole of the histidine or the NH function of arginine, benzyloxycarbonyl for the NH 2 function of lysine, formyl for the indole function of tryptophan;  15. Process for the in vitro detection of an infection with an HIV retrovirus capable of causing ALS or AIDS, characterized in that it comprises  a / bringing at least one peptide according to any one of Claims 1 to 6 into contact with a biological sample to be tested, capable of containing antibodies against the F protein of an HIV retrovirus, under conditions allowing formation an immunological peptide-antibody complex,  b / the detection of the immunological complex formed.  16. The detection method according to claim 14, characterized in that the peptide used specifically recognizes antibodies formed against the F protein of an HIV-1 retrovirus.  17. The detection method according to claim 15, characterized in that the peptide used specifically recognizes antibodies formed against the F protein of an HIV-2 retrovirus.  18. The method of claim 15, characterized in that it implements a mixture of peptides, capable of detecting an infection with a retrovirus HIV-1 or HIV-2.