DK174705B1 - Peptides having immunological properties corresponding to HIV-2 viruses, antigens and immunogenic agents containing such peptides, and methods and kits for an in vitro diagnosis of HIV-2 - Google Patents

Abstract

Peptides having immunological properties in common with those of the peptidic skeleton of peptides of viruses of the family HIV-2, particularly the envelope glycoprotein of HIV-2, characterized in that they have also a peptidic structure in common with the peptidic skeleton of peptides of SIV, particularly the envelope glycoprotein of SIV. The invention also relates to diagnosis compositions capable of detecting an infection due to HIV-2 and to vaccine compositions.

Description

DK 174705 B1

The present invention relates to peptides with immunological properties, possibly immunogenic properties, which are common to purified antigens obtainable from viruses capable of provoking lymphadenopathies which can develop into acquired immune deficiency syndrome. (AIDS) in humans.

More particularly, the invention relates to novel peptides of the kind set forth in claim 1. These peptides can be recognized by antibodies induced in humans by virus designated by the abbreviation HIV (according to nomenclature 10 defined in NATURE). The peptides of the invention have immunogenic properties can be made immunogenic in vivo, which immunogenicity can be manifested by an in vivo induction of antibodies that recognize the antigens characteristic of the HIV-2 virus, as well as (in respect of some of these peptides). antigens characteristic of HIV-1. The invention further relates to an antigenic agent of the kind set forth in claim 23 or an immunogenic agent of the kind set forth in claim 24.

The invention also relates to the use of these peptides for the in vitro diagnosis of HIV-2 infection in a biological fluid.

20

Finally, the invention relates to a kit for in vitro diagnosis of HIV-2 infection in a biological fluid.

Some of the present peptides can be used in in vivo methods of diagnosis of certain forms of AIDS in humans, and the peptides can also be used in systems or "kits" for diagnosis.

A first retrovirus, designated LAV-1 or HIV-1, has been isolated and described in GB Patent Application No. 83 / 24,800 and EP Patent Application No. No. 84 / 401,834 filed September 14, 1984. This virus has also been described. by F. Barre Sinoussi et al in Science 220, 45-99, pages 868-871.

2 DK 174705 B1

Variants of this HIV-1 virus, designated LAV, EU and LAV MAL, have also been isolated, characterized and described in EP Patent Application No. 84 / 401,834.

5 HIV-1 viruses and their variants have the following properties:

As preferred target cells, they have human Leu3 cells (or T4 lymphocytes) and their "immortalized" cell derivatives.

They have a reverse transcriptase activity which necessitates the presence of Mg 2+ ions, and exhibit a strong activity against poly (adenylate oligodoxydymidylase) poly (A) oligo (dT) 12-18).

| They have a density of 1.16-1.17 determined in a sucrose gradient.

15

They have a mean diameter of 139 nm and an average core diameter of 41 nm.

The lysates of these viruses contain a protein called p25 (core protein) which does not exhibit immunological cross-linking with the protein p24 from HTLV-1.

They contain a protein called p42 which belongs to their sheath, and they also contain a sheath glycoprotein called gp110 which has a molecular weight of 110,000.

25

The isolation and characterization of retroviruses belonging to a particular class and which have only a reduced immunological relationship with their origin are described in EP Patent Application No. 87 / 400,151.4. These retroviruses, which have been grouped under the designation HIV-2, have been isolated in connection with numerous African diseases where symptoms of a lymphadenopathy or an AlDS case occur.

3 DK 174705 B1

HIV-2 retroviruses, like HIV-1 retroviruses, are distinguished by a tropism against human T4 lymphocytes and by a cytopathogenic effect on these lymphocytes as they multiply. This causes both generalized and persistent polyadenopathies and AIDS.

In particular, it can be said that retroviruses purified from HIV-2 generally possess the following properties: the preferred target cells for HIV-2 retroviruses are human Leu3 cells (or 10 T4 lymphocytes) and immortalized cells derived from these T4 lymphocytes; they are cytotoxic to human T4 lymphocytes, they have a reverse transcriptase activity which necessitates the presence of Mg2 + ions and exhibits potent activity against poly (adenylate oligodeoxythymidylase) 15 (poly (A) oligo (dT) 12-18); In they have a density of 1.16 determined in a sucrose gradient; they have a mean diameter of 140 nm and a core with a mean diameter 20 of 41 nm; they can be grown in permanent strains of the HUT type or which express the T4 protein; They do not infect T8 lymphocytes; lysates of these viruses contain a protein called p26 that does not exhibit immunological cross-over with protein p24 from HTLV-I virus or HTLV-II virus; These lysates further contain a protein called p16 which is not immunologically recognized by the protein p19 of HTLV-I or HTLV-I I virus in radioimmunoprecipitation assay; 4 DK 174705 B1 I.

they also contain a molecular weight envelope glycoprotein of the order of 130,000-140,000 which does not exhibit immunological cross-over with gp110 from HIV-1 but which in turn crosses immunologically 5 with the envelope glycoprotein gp140 from STLV-III (virus isolated from monkeys); these lysates further contain 35S cysteine-tagged antigens whose molecular weight is between 32,000 and 42,000-45,000: they further comprise a molecular weight antigen of the order of 36,000 and an antigen of molecular weight of the order of 42,000, since one of these antigens (designated p36 and p42) is likely to be a transmembrane glycoprotein of HIV-2 virus, the genomic RNA of HIV-2 does not hybridize with genomic RNA of HIV-15 1 under stringent conditions; under non-stringent conditions, the HIV-2 genomic RNA does not hybridize with either the env gene or the LTR adjacent to the gene in HIV-1, or with sequences from the pol region of the HIV-1 genome; 20 under non-stringent conditions, it weakly hybridizes with the nucleotide sequences of the HIV-1 region.

Another retrovirus called SIV-1, which replaces the earlier term STLV-1H, is isolated from rhesus monkeys (MD Daniel et al. Science 228, 1201 (1985) NL Letwin et al., Science 230, 71 (1985). ) under the designation "STLV-lllmac").

A further retrovirus named "STLV-IIIagm" (or SIVagm) is isolated from wild green monkeys. But unlike the viruses present in the rhesus monkey, the presence of "STLV-IIIagm" does not appear to induce AIDS-type diseases in African green monkeys.

5 DK 174705 B1

A strain of retrovirus SIV-1mac was deposited at CNCM on February 7, 1986, under Nos. 1-521. Studies have shown that retrovirus SIV-1 contains certain proteins that exhibit a certain immunological relationship to structure-in proteins or glycoproteins obtainable under analogous conditions! 5 from HIV-2. This SIV-1 retrovirus, whose infectious nature has been found in monkeys, was named STLVIII by the researchers who isolated it (see the bibliographic references above).

For linguistic reasons, these viruses are hereinafter referred to only by the expression SIV (the term SIV is the English abbreviation for "Simian Immunodeficiency Virus"), optionally followed by an abbreviation indicating the monkey from which they were obtained. , such as MAC or mac for rhesus monkeys or AGM for African green monkeys (abbreviation for African Green Monkey).

15

Using the same techniques as described above, it has been found that one can also obtain from SIV-1 mac: a principal nuclear protein called p27, having a molecular weight of the order of 27 kd, a larger envelope glycoprotein, called gp140, a protein called p32, which is probably a transmembrane protein, is rarely observed by radioimmunoprecipitation assay (RIPA) when the virus is previously labeled with 35S-cysteine, but which can be observed in broad-band immunoassay samples.

More precise studies have been carried out on the above viruses HIV-30 2 and SIV virus. Continued studies of HIV-2 retroviruses have also led to the generation of complementary DNA sequences (cDNA) for RNA in their genomes. The complete nucleotide sequence of cDNA for a representative HIV-2 (HIV-2-ROD) retrovirus was deposited on February 21, 2006 by CNCM under I-522 under the reference designation LAV-II ROD.

This nucleotide sequence and the open reading frames that they contain are given in Figure 1A.

Furthermore, continued studies of other retroviruses have also enabled the mapping of their complete nucleotide sequences. This is especially true for cDNA derived from genomic RNA from SIV virus.

10

Cloning and sequencing of SIV-1mac virus enabled mapping of its nucleotide sequence and was performed under the following conditions: 15 DNA from HUT 78 cells infected with SIV virus (isolate STLV-III mac 142-83 described by Daniel et al (1985) Science, 228 pages 1201-1204), and partially digested with the restriction enzyme Sau3A was cloned at the BamHI restriction site in the bacteriophage vector ELBL3 to obtain a genomic library. The 2 million recombinant phages in the thus compiled genomic library were examined in situ under safety assays P3 using sequences of HIV-2 virus derived from clones lambda-ROD4, lambda-ROD35 and E2 (Clevel et al (1986) Nature , 324. page 691) and was nick-translated.

The hybridization was performed at 5xSSC at 50 ° C and the leaches at 2xSSC at 50 ° C. A single clone containing all virus sequences was obtained. This clone was named lambda-SIV-1. The insert in the phag lambda-SIV-1 measures 16.5 kb in total and includes an integrated provirus, where only the first 250 bases are missing from the left-sided LTR, while the 30 right-sided LTR is complete.

7 DK 174705 B1

The integrated provirus was sequenced by the dideoxynucleotide method after subcloning random fragments into the pha gene M13mp8. 300 subclones were analyzed.

In '5 cDNA fragments derived from the clone lambda-SIV-1 inserted into plasmids pSIV1.1 and pSIV-1.2 were deposited by CNCM on April 15, 1987 under numbers I-658 (pSIV-1.1) and I-659 ( pSIV-1.2).

The results are given in the figures described below.

10

FIG. TB denotes the nucleotide sequence of the genome from SIV virus and the sequences derived therefrom for the viral proteins corresponding to the products of the genes gag, pol, env, Q, X, R, tat, species, F.

FIG. 3-11 and FIGS. Figure 1C shows comparison of the theoretical products of the viral genes and of the LTR between HIV-2 and SIVmac (_SIV-1).

The nucleic acid sequences of cDNA from SIV are positioned against the nucleic acid sequences of the HIV-2 ROD virus as far as the LTR sequence is concerned (cf. FIG.

20 ° C). This presentation as recognized from the complete genome by comparing FIG. 1B with FIG. 3-11 enables one to find or deduce the nucleic acids that have essential structural elements in common with the two viruses.

Comparative studies which have also enabled results to be obtained for nuclear proteins, hereinafter referred to as gag proteins, as well as envelope proteins, hereinafter referred to as env proteins, are also described in the above-mentioned EP Application No. 87 / 400,151.4. . These results show that nuclear proteins (gag proteins) in HIV-2 exhibit less distinct differences from 30 cells of virus HIV-1 than the envelope proteins (env proteins). Overall, the env proteins in HIV-2 have been shown to exhibit extremely weak, non-existent immunological similarities to the corresponding env proteins in the HIV-1 virus.

8 DK 174705 B1

By contrast, comparative studies between the structures of the cDNA sequences in HIV-2 and SIV virus have enabled the identification of certain common characteristics that appear at the protein level.

5

Overall, the proteins from HIV-2 and SIV-1 exhibit important immunological similarities.

The major envelope protein of HIV-2 has been found to be more closely related to the major envelope glycoprotein of SIV in immunological terms than to the essential envelope glycoprotein of HIV-1.

These findings are not only apparent in terms of molecular weights: 130-140 kd for the major HIV-2 and SIV glycoproteins versus ca. 110 15 kd for the major envelope glycoprotein of HIV-1, but also in terms of its immunological properties, since sampled serum samples from patients infected with HIV-2 and especially antibodies produced against gp140 from HIV-2 recognize gp140 from SIV-1 mac, while the same serum samples and the same HIV-2 antibodies in similar experiments do not recognize gp110 from 20 HIV-1. But anti-HIV-1 serum, which has never reacted with gp140 from HIV-2, precipitates a 26 kd protein labeled with 35S-cysteine contained in HIV-2 extracts.

The major core protein from HIV-2 appears to exhibit an average molecular weight of approx. 26,000, which is between the molecular weight of p25 from HIV-1 and p27 from SIV.

These observations stem from trials conducted on virus extracts obtained from HIV-2 isolated from one of the above patients. Similar results have been obtained with viral extracts of HIV-2 isolated from another patient.

9 DK 174705 B1

More extensive experiments have led the inventors to recognize a first class of peptides having amino acid sequences that are either identical or closely related to sequences contained within the interior of the structures of the gag proteins for HIV-2 or from SIV, and even from HIV. -1. These pep-5 times are particularly useful for diagnosing an infection in humans | with HIV-2 virus or a variant thereof.

| Against this background, the method and agent of the invention can be used to diagnose in vitro antibodies against HIV-2 viruses or variants thereof, especially in biological samples, especially serum samples from patients who have been infected with HIV infection. 2 viruses, some of which in! peptides allow a particularly clear discrimination between infections caused by HIV-2 virus and HIV-1 virus.

These extensive studies have also led to the possibility of synthesizing peptides that are immunogenic or can be immunogenic and which exhibit structural characteristics that enable them to induce in vivo the formation of antibodies capable of recognizing env proteins. both from HIV-1 and HIV-2, and at least for some of these peptides to bind both to HIV-1 virus and to HIV-2 virus, specifically for the purpose of neutralizing them. Thus, the use of these types of peptides is particularly indicated for the preparation of active ingredients of vaccines against HIV virus, and thus against AIDS.

To describe below the amino acid residues included in the structure of the peptides, one uses for the amino acid the unique international nomenclature which denotes each natural amino acid with a capital letter according to the table below: 30 M Methionine L Leucine I Isoleucine V Valin DK 174705 B1 i 10 i F Phenylalanine S Serine P Prolin T Threonine 5 A Alanine Y Tyrosine IH Histidine | Q Glutamine 10 N Asparagine K Lysine D Aspartic acid E Glutamic acid C Cysteine 15 W Tryptophan R Arginine G Glycine When an amino acid p.g.a. its position in the middle of the amino acid chain characteristic of a given peptide may have several meanings, it may either be denoted by a hyphen if its meaning may be arbitrary, or by a lower case when that amino acid may exhibit a number preferred meanings, however, and this number is always greater than 1. In the latter case, the possible meanings of the lowercase letter are always given in connection with the peptide to which it belongs.

To facilitate reading, these peptides are also denoted by an abbreviation env followed by a number, by reference to the contained amino acids, as appropriate, in the env proteins of certain types of HIV-1, HIV-2 or 30 SIV. This is discussed in more detail below.

In the following definitions, the groups X denote either a free NH 1 group or an amidated group especially with one or two alkyl groups containing 1 -5 carbon atoms, or a peptide group containing 1 -5 amino acids, wherein the N-terminal amino acid itself exhibits a free NH 2 group or amide group, as indicated previously, and groups Z denote either a free -OH group or alkoxy group and then contain an alkyl group comprising 1-5 carbon atoms, or a peptide group comprising 1-5 amino acids if C-terminal amino acid itself exhibits a free -OH group or alkoxy group, as indicated above, the groups of 1-5 amino acids optionally included in X or Z or both being those whose presence is incompatible with conservation essentially the immunological, possibly immunogenic properties of the peptides from which they are removed.

The peptides of the invention, which have immunological properties in common with antigens from HIV-2 and, for some of them, furthermore with antigens from HIV-1 or variants thereof, are characterized in that they also have a peptide structure in common with antigens from SIV. Advantageously, these peptides usually comprise a maximum of 40 amino acid residues.

Preferred peptides are the following: 20 env1

XRV-AIEKYL-DQA-LN-WGCAFRQVCZ

env2 25 X-LE-AQI-QQEKNMYELQKLNZ env3

XELGDYKLVEITPIG-APT-KR ---- Z

30 env4

X — VTV-Y G VP-WK-AT-LF C A-Z

, DK 174705 B1 12 env5 X — QE-L-NVTE-F-W-NZ env6

5 XL — S-KPCVKLTPLCV - Z

env7

| X-N-S-IT - C-C-Z

Env8 X-1 — YC-P-G-A-L-C-N-TZ env9

X ----- ----- A-C, W-Z

15

env1Q

X-G-DPE-NC --- GEF-YCN ----- NZ

env11

X-C-IKQ-I ---- G-YZ

More specifically, the invention relates to the following peptides: env1 XRV-AIEKYL-DQA-LN-WGCAFRQVCZ env2 X-LE-AQIQQEKNMYELQKLNZ env3

XELGDYKLVEITPIG-APT-KR ---- Z

env4 13 DK 174705 B1 X — VTV-YGVP-W-AT-LF CA-Z env5 X — E - L-NVTE-FW-NZ 5 env6 XL — S-KPCVKL-PLC — Z env7 10 X — NS1— CK —Z env8

X-L-YC-P-G-A-L-C-N-TZ

15 env9

X ---- C ---- A-W-Z

env10

Z-G ---- DPE-NC-GEF-VC --- NZ

20 env11

X-C-L-Q-L-G ---- YZ

Particularly advantageous peptides similar to the above exhibit the following formulas: env1 XRVTAIEKYLQDQARLNSWGCAFRQVCZ, or XRVTAIEKYLKDQAQLNAWGCAFRQVCZ 30 env2

XSLEQAQIQQEKNMYELQKLNSWZ, or XLLEEAQIQQEKNMYELQKLNSWZ

14 DK 174705 B1 env3! XELGDYKLVEITPIGFAPTKEKRYSSAHZ, or

5 XELGDYKLVEITPIGLAPTNVKRYTTG-Z

It is noted that the peptides env1. env2. env3 confirms the very similarity between HIV-2 and SIV-1. In fact, the first peptide is included in the HIV-2 genome and the second in the SIV-1 genome.

10 env4

XabsdVTVeYGVPfWogAThiLFCAjZ, in which the letters a-j and o can have the following meanings: 15

a is C, E or D b is T, K, D, N or I c is Q or L d is Y or W 20 e is F or Y f is T, V or A g is N or E h is I or T i is P or T 25 j is T or S o is K or R

env5

XabcoEdeLfNVTEgFhiWjNZ, 30

wherein the letters a-j and o can have the following meanings: a is D or P

15 DK 174705 B1

b is D or N c is Y or P

d is I, V, I or L 5 e is T, V, E or A f is V, G or E or -g is A, N, G or S h is D or N i is A or M 10 j is N, K or E o are Q or S

env6 XLabcSd KPCVKLoP LCuef KZ, 15 in which the letters a-f, o and u can have the following meanings:

a is F or W b is E or D 20 c is T or Q d is I or L e is A, S or T f is M or L o is T or S 25 u is V or I

env7

XabCNxSylocdCeKfghiZ, 30 in which the letters a-i and x and y can have the following meanings:

a is N or T or I b is H or S or N

16 DK 174705 B1

c is E or Q d is S, A or C

e is D or P 5 f is H, Veller D g is Y or S h is W or F i is D or E X is T or R 10 y is Veller A

0 is T or Q

env8

XalbcdYCxPeGfAgLhCiNjTZ, wherein the letters a-k and x can have the following meanings:

a is A or P b is R or P 20 c is F, I or C d is R or H e is P or A f is Y or F g is L or I 25 h is R or K

1 is - or N

j is D or K x is A or T

30 env9

XwabcxyAdCefghizWjkZ, in which the letters a-k and x-z can have the following meanings: 17 DK 174705 B1

a is K or - or E b is R or -5 c is P or M or I d is Weller H or Y e is W or N or T or R f is F or I

g is K or S or N or G 10 h is G or R or E i is - or A or T j is K or N or D or S k is D or A or N or K or E w is N, D or In 15 x, R or G or K y is Q or K or R z is K or E or Q or N

env10 20 XaGbDPEcdefghNCiGEFjYCokxImnNZ, in which the letters a-n and x can have the following meanings:

a is K or - or G 25 b is S or G or -c is V or I d is A or V or T e is Y or T or M or F f is M or H 30 g is W or S h isT or F i is R or G j is L or F

18 DK 174705 B1

0 is N or K k is M or S

1 is W or Q or K or G 5 m is F or L

n is L or F x is T or S or N

in env11 10 XabcdwCeloQflxgyhizGjklYZ, in which the letters a-l and w-z can have the following meanings:

a is R or T or S or N 15 b is N or I c is Y or T d is A or L or V e is H or R

in f is I or F

20 g is T or M

h is H or Q or A i is K or E j is R or K k is N or A 25 I is V or M w is P or Q x is N or K y is W or V z is V or T or K O is K or R

It should generally be noted that the amino acids which have a unique meaning (and thus represented by a capital letter corresponding to the international nomenclature) and which are included in the definitions of the peptides of the invention set forth above, are found to correspond to identical amino acids located in the same order in the corresponding sequences of at least one HIV or SIV-1 virus.

5

The positions of these sequences are underlined and distributed within the amino acid sequences of the eny proteins for HIV-2 ROD (CNCM No. I-532) and HIV-1 BRU (CNCM No. I-232), respectively, shown in FIG. 2. The order of the amino acids in the env and gag proteins for SIV-1 mac, respectively (CNCM no.

10-1-521) and of the HIV-2 ROD shown in FIG. 3 and FIG. 4th

The unbroken lines shown at certain sites of these sequences should emphasize that certain amino acids contained in these sequences are voluntarily omitted in the presentation to allow identical amino acids to be aligned (marked with an asterisk) or vertical points on one and the same vertical line in the corresponding protein sequences from on the one hand HIV-1 and HIV-2 and on the other hand SIV and HIV-2.

Apart from the above peptides, the invention also relates to peptides modified by insertion and / or deletion and / or substitution of one or more amino acids, as long as the antigenic or immunogenic properties of these peptides are not modified, or that the recognition properties of the antigen or antibody are of these peptides are not substantially modified.

In a particularly preferred embodiment, the invention relates to peptides that have immunological properties in common with the peptide backbone of the HIV-2 class virus glycoprotein, these peptides containing a number of amino acid residues not exceeding 40.

30

These preferred peptides of the invention exhibit the following sequences: env1

RVTAIEKYLQDQARLNSWGCAFRQVC

AIEKYLQDQ

RVSAIEKYLKDQAQLNAWGCAFRQVC

AIEKYLKDQ

5 env2

SLEQAQIQQEKNMVELQKLNSW

QIQQEKN

LLEEAQIQQEKNMYELQKLNSW

10 env3

ELGDYKLVEITPIGFAPTKEKRYSSAH

YKLVEITPIGFAPTKEK

ELGDYKLVEITPIGLAPTNVKRYTTG-

15 YKLVEITPIGLAPTNVK

env4

CTQYVTVFYGVPTWKNATIPLFCAT VTVF Y G VPTWKN AT

20 CIQYVTVFYGVPAWRNATIPLFCAT VTVFYGVPAWRNAT

EKLWVTVYYGVPVWKEATTTLFCAS VTVYY G VP VWKE AT

25 E DL WVTVYY G VP VWKEATTTLF C AS VTVYY G VP VWKE AT

D N L WVTVYY G VP VWKEATTTLFC AS VTVYYGVPVWKEAT

30 env5

DDYQEITL-NVTEAFDAWNN

L-NVTEAF

21 DK 174705 B1

DDYSELAL-NVTESFDAWEN

L-NVTESF

PNPQEWLVNVTENFNMWKN

LVNVTENF

5 PNPQEIELENVTEGFNMWKN LENVTEGF

PNPQEIALENVTENFNMWKN

LENVTENF

10 env6

ETSIKPCVKLTPLCVAMK ETSIKPCVKLSPLCITMR DQSLKPCVKLTPLCVSLK DQSLKPCVKLTPLCVTLN 15 PCVKLTPLCV

env7

NHCNTSVITESCD

NTSVIT

20 NHCNTSVIQECCD NTSVIQ

TSCNTSVITQACP

NTSVIT

INCNTSVITQACP 25 NTSVIT

INCNTSAITQACP

NTSAIT

env8

30 YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT YCAPAGFAILKCNDKK YCAPAGFAILKCRDKK

22 DK 174705 B1 env9

NKRPRQAWCWFKG-KWKD NERPKQAWCRFGG-NWKE 5 N-MRQAHCNISRAKWNA D-IRRAYCTINETEWDK l-IGQAHCNISRAQWSK

env10

10 KGSDPEVAYMWTNCRGEFLYCNMTWFLN NCRGEFLYCN

GG-DPEVTFMWTNCRGEFLYCKMNWFLN

NCRGEFLYCK

-GGDPEIVTHSFNCGGEFFYCNSTQLFN 15 NCGGEFFYCN

-GGDPEITTHSFNCRGEFFYCNTSKLFN

NCRGEFFYCN

-GGDPEITTHSFNCGGEFFYCNTSGLFN

NCGGEFFYCN

20 env11

RNYAPCHIKQIINTWHKVGRNVY

CHIKQII

RNYVPCHIRQIINTWHKVGKNVY 25 CHIRQII

TITLPCRIKQFINMWQEVGKAMY

CRIKQFI

SITLPCRIKQIINMWQKTCKAMY 30 CRIKQII

NITLQCRIKQIIKMVAGR-Kaiy

CRIKQII

23 DK 174705 B1

The peptides of the invention can advantageously be prepared by the classical methods of peptide synthesis. This synthesis can be carried out in homogeneous solution or in solid phase.

For example. For example, the homogeneous solution synthesis technique described by HOUBEN-WEYL in "Methode der Organischen Chemie", Ed. E. Wunsch, Vols 15-1 and II., THIEME, Stuttgart 1974.

In this method, two and two successive 10 aminoacyl groups are successively condensed in the required order or condensed aminoacyl groups and pre-formed fragments which already contain several aminoacyl groups in the correct order, or more thus previously prepared fragments, it being understood that one has before taken! to protect all the reactive groups on these aminoacyl groups or fragments, except the amino groups in one and the carboxyl groups in the other or vice versa, which should normally be included in the formation of peptide bonds, especially after activation of the carboxyl group by well known methods in peptide synthesis. As a variant, coupling reactions can be utilized using classical coupling agents of the carbodiimide type, such as e.g. 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide.

When the aminoacyl group used exhibits an additional acidic function, especially in the case of glutamic acid, these may be groups protected, e.g. with t-butyl ester groups.

For progressive synthesizing amino acid after amino acid, the synthesis preferably begins with condensation of the C-terminal amino acid with the amino acid corresponding to the adjacent aminoacyl in the desired sequence and then still up to the N-terminal amino acid. In the second preferred technique according to the invention, the method described by 30 R.D. Merrifield, "Solid phase peptide synthesis" (J. Am. Soc., 45, 2149-2154).

24 DK 174705 B1 i

To prepare a peptide chain according to the Merrifield method, a very porous polymeric resin is used to fix the first C-terminal amino acid in the chain. This amino acid is fixed to the resin by its carboxyl group while its amino group is protected, e.g.

5 with a t-butyloxycarbonyl group.

Thus, when the first C-terminal amino acid is fixed on the resin, the protecting group on the amino group is removed and by washing the resin with an acid.

When the amino group protecting group is t-butyloxycarbonyl, it can be removed by treating the resin with trifluoroacetic acid.

The second amino acid which delivers the second aminoacyl in the desired sequence from the C-terminal aminoacyl is then coupled to the de-protected amino group of the first C-terminal amino acid fixed to the resin.

Preferably, the carboxyl group of the second amino acid is activated, e.g. with dicyclohexylcarbodiimide and the amino group is protected, e.g. with t-butyloxycarbonyl.

Thus, the first portion of the desired peptide chain comprising two amino acids and whose terminal amino group is protected is obtained. As before, the amino group is deprotected and it is then possible to proceed with fixation of the third aminoacyl under analogous conditions to those used in the addition of the second C-terminal amino acid.

Thus, one after the other of the amino acids which are to form the peptide chain is fixed to the amino group, each time previously de-protected, to the already formed part of the peptide chain which is bound to the resin.

When the entire desired peptide chain is formed, the protecting groups of the various amino acids constituting the peptide chain are removed and the peptide is released from the resin, e.g. using hydrofluoric acid.

The invention also relates to water-soluble oligomers of the above-mentioned monomeric peptides. The oligomerization can induce an increase in the immunogenicity of the monomeric peptides of the invention. Without limiting this number, it can nevertheless be mentioned that these oligomers e.g. may contain 2-10 monomers-10 moieties.

The monomer units included in this oligomer are either all of the polypeptide of sequence 1 or of the polypeptide of sequence 2 or of one or the other of these polypeptides.

15

Also, to carry out the oligomerization, any conventional polymerization technique within the peptide chemistry can be employed, this polymerization being carried out until an oligomer or polymer is obtained which | contains the required number of monomeric units to achieve the desired immunogenicity.

One method of oligomerization or polymerization of the monomer consists in reacting it with a crosslinking agent such as glutaraldehyde.

Other oligomerization or coupling methods, e.g. the method utilizing successive couplings of the monomer units by their terminal carboxyl and amino groups in the presence of bifunctional homo or heterocoupling agents.

Also, for the production of molecules comprising one or more units of the above 17 amino acids, the genetic engineering methods utilizing microorganisms transformed with a given nucleic acid which comprise the appropriate corresponding nucleotide sequences can be used.

In the invention, nucleic acids are provided which contain one or more sequences derived from the cDNA sequence of HIV-2 ROD virus. These sequences, which are marked by the numbering which appears on the sequence described above, encode certain interesting peptides of the invention.

Sequence encoding env1 nucleotide 7850 - 7927 - - - env2 - 8030-8095 - - - env3 - 7601 -7636 - - - env4 - 6170-6247 - - - env5 - 6294-6349 15 - - - - env6 - 6392 - 6445. . . env7 - 6724 - 6763 - - - env8 - 6794-6838 - - - env9 - 7112-7162 - - - env10-7253-7336 20 - --- env11-7358-7426

The corresponding nucleic acids from SIV virus also contain one or more sequences derived from cDNA from SIV-1 virus. These sequences encoding the env1-env11 peptides can be located in FIG. 3 by comparison with the corresponding sequences described for HIV-2.

When the immunogenic agents are conjugated to a carrier molecule as claimed in claim 24, this is accomplished by covalently coupling the peptides of the invention (or said oligomers) to natural or synthetic physiologically acceptable and nontoxic carrier molecules by complementary reactive groups which are carried by the carrier molecule and the peptide, respectively.

Examples of suitable groups are illustrated in the following: in 27 DK 174705 B1

As examples of carrier molecules or macromolecular carriers that! may be included in the constitution of conjugates according to the invention, one may mention natural proteins such as tetanus anatoxin, ovalbumin, serum albumin, hemocyamines etc.

5

As examples of synthetic carrier molecules, e.g. mention poly-lysines or poly (D-L-alanine) -poly (L-lysine).

In the literature, other types of useful macromolecular carriers are mentioned, 10 and these generally exhibit a molecular weight in excess of 20,000.

In order to synthesize the conjugates according to the invention, methods known per se can be used, e.g. as described by FRANTZ and ROBERTSON, Infect, and Immunity, 33, 193-198 (1981), or P.E. KAUFFMAN, 15 Applied and Environmental Microbiology, (October 1981), Vol. 42, No. 4, 611-614 using an appropriate peptide and carrier molecule.

In practice, it is advantageously used as a coupling means, e.g. the following compounds: glutaraldehyde, ethyl chloroformate, water-soluble carbodiimides, such as N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide, HCl, diisocyanates, bis-diazobenzidine, di- and trichloro-s-triazines, cyanogen bromide and the coupling means mentioned in Scand. J. Immunol. (1978), Vol. 8, pp. 7-23 AVRAMEAS, TERNYNCK, GUESDON).

Any coupling process using one or more reactive groups in the peptide and one or more reactive groups in the carrier molecules can be used. Advantageously, these are carboxyl and amino groups which may give rise to a coupling reaction in the presence of a coupling agent of the type used in protein synthesis, e.g. 1-30 Ethyl 3- (3-dimethylaminopropyl) carbodiimide, N-hydroxybenzotriazole, etc.

Glutaraldehyde can also be used, especially when it comes to interconnecting amino groups sitting on the peptide and the carrier molecule, respectively.

28 DK 174705 B1

The peptides of the invention exhibit antigenic properties. Thus, they can be used in diagnostic methods to detect an infection with HIV-2 virus.

5

As mentioned earlier, studies have made it possible to distinguish between two groups of peptides that can be used in methods for detecting antibodies to HIV-2 virus in human biological fluids, especially serum or cephalospinal fluid.

10

One group comprises peptides which are more specifically similar to those peptides located in the transmembrane portion and at the end of the outer portion of the envelope protein. These proteins are referred to above as env1. env2 and env3. They enable a specific recognition of the presence of antibodies against HIV-2 and thus enable discrimination between the above! or infections present due to HIV in a person, more specifically a discrimination between infections provoked by HIV-2 and infections provoked by HIV-1.

The invention also relates to an antigenic agent containing at least one of the above peptides or at least one oligomer of such peptide, which is characterized in that it has the ability to be recognized by human origin serum containing HIV antibodies. 2nd

The invention further relates to a method for in vitro diagnosing one or more peptides of the invention in order to detect antibodies to HIV-2 in biological fluids, especially human serum.

More generally, the in vitro diagnostic method comprises the following steps:

The biological fluid is contacted with the peptides, in the presence of a peptide-antibody complex, if any, is detected by physical or chemical methods in the biological fluid.

In a preferred embodiment of the method of the invention 5, the detection of the antigen-antibody complex is performed by immunoenzymatic (ELISA type), immunofluorescence methods (IFA type), radioimmunological methods (RIA type) or radioimmunoprecipitation methods (type RIPA). Thus, the invention also relates to any peptide of the invention labeled by a suitable marker, e.g. an enzymatic, fluorescent or radioactive marker.

Such methods include e.g. the following steps: depositing determined amounts of a peptide-containing agent of the invention into the wells of a microtiter plate, placing increasing dilutions of the serum to be diagnosed in the wells, incubating the microtiter plate and repeated rinses thereof, introducing into the wells labeled antibody to the immunoglobulins in the blood, wherein the labeling of these antibodies may be accomplished by an enzyme selected from enzymes capable of hydrolyzing a substrate by modifying the absorption of its radiation, at least at a determined wavelength, detection by comparison with a control sample of the hydrolyzed 30 amount of substrate.

The invention also relates to in vitro diagnostic kits for the presence of antibodies to HIV-2 virus in a biological medium which comprises: reagents for providing a composition of the medium which is suitable for carrying out the immunological reaction, reagents which enable detection of the antigen-antibody complex are formed by the immunological reaction. Such reagents may also be provided with a marker or be capable of being recognized by a labeled reagent. This is especially the case when the above peptidic agent is not labeled.

- a biological reference tissue fluid free of antibodies recognized by the above peptidic agent.

15

Antibodies to the peptides of the invention can be formed and this production is not limited to polyclonal antibodies.

It also finds application to any monoclonal antibody produced by any hybridoma that can be formed by classical methods from spleen cells of an animal, especially mice or rats immunized with one of the peptides of the invention, and cells from a suitable myeloid cell line that can be selected for its ability to produce monoclonal antibodies that recognize the peptide originally used for the immunization of the animal.

The invention further relates to immunogenic agents for the preparation of vaccines whose active ingredient consists of at least one peptide of the invention or an oligomer of such peptide or peptide conjugated to a carrier molecule which is peculiar in that it induces the formation of antibodies against these peptides in sufficient quantity to also inhibit HIV-2 retrovirus proteins, even HIV-2 retroviruses themselves, in association with a pharmaceutically acceptable carrier.

In DK 174705 B1 in 31

Specifically, the immunogenic agents for the preparation of vaccines comprise at least one of the peptides referred to above env4. env5. env6. env7. env8. env9. env10. envelope or mixtures thereof. Among these five peptides, which are suitable for constituting active ingredients in vaccines, some are particularly preferred because they exhibit a basic amino acid structure corresponding to regions of envelope glycoproteins exhibiting a high degree of conservation, not only in HIV. 2 and in SIV, but also in HIV-1 virus. These peptides, which are particularly preferred, are those peptides designated env4. and certain 10 of the peptides env5. env6 and env10.

In a preferred embodiment of the invention, the immunogenic peptides or fragments thereof which are suitable as active ingredients in vaccines are selected from the formulas corresponding to the sequences exhibited in the envelope glycoproteins of HIV-2, SIV and HIV-1. an amino acid homology exceeding 50%, which belongs to the external part of the envelope of viruses, which is free or almost free of deletions, and which contains cysteine residues which promote the stability of the bonds and the formation of "hairpins".

The following peptides belong to this category of preferred peptides.

env4

XVTV-YGVP-W-ATZ

25 env5

XL NVTE-FZ

env6

XKPCVKL-PLC-Z

30 env7

XN-S-I-Z

32 DK 174705 B1 env10

XNC-GEF-YC-Z

env11

5 XC-I-Q-IZ

Advantageous pharmaceutical agents consist of injectable solutions, suspensions or liposomes containing an effective dose of at least one product of the invention. Preferably, these solutions, suspensions or liposomes are provided in a sterile isotonic aqueous phase, preferably a physiological saline or glucose solution.

Specifically, such suspensions, solutions or liposomes are capable of being administered by intradermal, intramuscular or subcutaneous injection or by co-administration.

Pharmaceutical agents may also be administered by other means, especially orally.

Pharmaceutical agents which can be used as vaccines may be effective in producing antibodies against HIV-2 virus, e.g. is administered at doses of 10-500 pg / kg of the peptides of the invention, preferably 50-100 pg / kg.

These doses are given by way of example only and should not be construed as limiting.

As noted above, the various more defined peptides may comprise modifications which do not have the effect of fundamentally modifying the immunological properties. The equivalent peptides thus obtained are covered by the following claims. As an example of equivalent peptides, mention may be made of peptides whose structures correspond to cDNA regions in other variants of HIV-2, SIV or HIV-1 when these regions are aligned under the conditions similar to those described above in 33 DK 174705 B1 association with HIV-2 ROD, SIV and HIV-1 BRU. As examples of other of these peptides, mention may be made of peptides whose structure corresponds to the regions of the cDNA deposited by CNCM, especially with numbers I-502, I-642 (HIC-2 IRMO) and I-643 (HIV 2 EHO) as well as, in some cases, variants of 5 HIV-1 deposited by CNCM with numbers I-232, I-240, 1-241, I-550 and 1-551.

The peptides of the invention may also be defined by the following formulas wherein X, Z and the hyphens have the above meanings:

XRV-AIEKYL-DQA-LN-WGCAFRQVCZ

XAIEKYL-DZ

15 X-LE-AQIQQEKNMYELOQKLNSWZ XQIQQEKNZ

XELGDYKLVEITPIG-APT-KR - Z

XYKLVEITPIG-APT-KRZ

20

X — VTV-Y G VP-W-AT - LF C A-Z XVTV-YGVP-W — ATZ

X — E - L-NVTE-F - W-NZ 25 XL-NVTE-FZ

XL — S-KPCVKL-PLC — Z XKPCVKL-PLC-Z XS-KPC VKL-P LC-Z 30

X — N-S-1 — C-Z XN-S-I-Z

34 DK 174705 B1

XYC-P-G-A-L-C-N-TZ

X ---- ----- A-C ~ W Z

5 NKRPRQAWCWFKG-KWKD

X-G ----- DPE-NC-GEF-YC ----- NZ

X-C-L-Q-L-G ---- YZ

10

The invention also relates, in addition to the already described SIV peptide proteins, which are encoded by cDNA from the SIV virus. They also apply to proteins of any immunological virus that are immunologically closely related to SIV-1 mac, especially any virus whose envelope proteins and - 15 glycoproteins exhibit immunological cross-over and whose cDNA exhibits a homology percentage of at least 95%, preferably most 98%.

In particular, the invention relates to envelope proteins and glycoproteins encoded by the env gene. and shown in FIG. 3, 1 20

The amino acids of the above SIV proteins are shown in line with the amino acid sequences of the corresponding proteins of the HIV-2 virus; the verti- | bald dots that appear between the two sequences correspond to amino acids,! common to the proteins of the two viruses.

The cDNA sequences encoding the aforementioned proteins are shown in FIG.

ΊΒ.

These cDNA sequences located by the numbering that appear on the previously described sequences (Figure 1B) are as follows: 30 - GAG Coding Sequence, Nucleotides 551-2068 - ........ POL, "1726-4893 - ........ Q, "4826-5467 35 DK 174705 B1 - ......" X, "5298-5633 - ........ R, * '5637-5939 | - ........ F, "8569-9354 5 - ........ TAT-1," 5788-6084 - ........ ART-1, "6014-6130 - ........ TAT-2, "8296-8391 - ........ ART-2," 8294-8548 - ........ ENV, "6090-8732 10

The invention thus, of course, relates to the previously described proteins, whether obtained from SIV virus or made from synthetic pathway, especially by the methods mentioned above in connection with the synthesis of smaller peptides.

15

The invention also relates to the use of the foregoing proteins for the diagnosis of the possible presence of antibodies directed against HIV-2 proteins, or even to all of HIV-2 or, for some of them, the use for the diagnosis of an infection due to an HIV virus. The proteins 20 ENV are preferably used for the specific diagnosis of an infection with HIV-2 or a variant thereof, and sometimes for the diagnosis of an infection of HIV-2 or HIV-1.

Thus, the invention also relates to an in vitro diagnostic method for detecting antibodies to HIV-2 and, optionally, to HIV-1 in biological fluids and especially in human serum. Such methods useful for using the above SIV proteins as diagnostic proteins have been described previously.

Thus, the invention also relates to kits for in vitro diagnosis of the presence of antibodies against HIV-2 virus and also in cases against HIV-1 virus in a biological medium. Such kits utilizing the above peptides are also described above.

36 DK 174705 B1

Furthermore, the invention relates to immunogenic agents for the production of vaccines whose active ingredient consists of at least a portion of the protein ENV from the SIV virus, this protein also being conjugated to a carrier molecule. These immunogenic agents induce the formation of antibodies against this peptide in sufficient quantity to inhibit proteins from HIV-2 retroviruses or even HIV-2 retroviruses themselves.

10 i j

Claims (29)

1. A peptide exhibiting immunological properties common to the peptide backbone of the envelope glycoprotein of the HIV-2 virus, characterized in that it also exhibits a peptide structure common to the peptide backbone of the glycoprotein of SIV-1 virus. A peptide according to claim 1, characterized in that the number of amino acids does not exceed 40. Peptide according to claim 2, characterized in that it has one of the general formulas: XRV-AIEKYL-DQA-LN-WGCAFRQVCZ XAIEKYL-DZ wherein X and Z are OH or NHr groups or when the immunological properties of peptides are liberated for these groups are not substantially modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the 20 hyphens represents an aminoacyl residue selected from those which enable the indicated peptide to retain the immunological properties of one of the following sequences: RVTAIEKYLQDQARLNSWGCAFRQVC 25 AIEKYLQDQ RVSAIEKYLKDQAQLNAWGCAFRQVC AIEKYLKDQ Peptide according to claim 2, characterized in that it has one of the general formulas: X-LE-AQIQQEKNMYELQKLNSWZ XQIQQEKNZ | DK 174705 B1 wherein X and Z are OH or NH2 groups, or when the immunological properties of peptides liberated from these groups are not significantly modified, | X and Z may denote groups comprising 1-5 amino acid residues, and each of! The dashes denote an aminoacyl residue selected from those which indicate that the specified peptide retains the immunological properties of one of the following sequences: SLEQAQIQQEKNMYELQKLNSW 10 QIQQEKN LLEEAQIQQEKNMYELQKLNSW 5 RNYAPCHIKQIINTWHKVGRNVY CHIKQII RNYVPCHIRQIINTWHKVGKNVY CHIRQII TITLPCRIKQFINMWQEVGKAMY 10 CRIKQFI SITLPCRIKQIINMWQKTCKAMY CRIKQtl NITLQCRIKQIK Peptide according to claim 2, characterized in that it has one of the general formulas: XELGDYKLVEITPIG-APT-KR ---- Z XYKLVEITPIG-APT-KRZ wherein X and Z are OH or NH 2 groups or immunological properties of peptides released for these groups are not substantially modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which allow the peptide to retain the immunological properties of one of the following sequences: 25 ELGDYKLVEITPIGFAPTKE KRYSSAH YKLVEITPIGFAPTKEK ELGDYKLVE ITPIGLAPTNVKRYTTG-YKLVEITPIGLAPTNVK 30 Peptide according to claim 2, characterized in that it has one of the general formulas: DK 174705 B1 X - VTV-YGVP-W-AT-LFCA-Z XVTV-Y GVP-W - ATZ wherein X and Z are OH or NH 2 groups, or when the immunological properties of peptides liberated from these groups are not significantly modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which enable the indicated peptide retains the immunological properties of one of the following sequences: 10 CTQYVTVFYGVPTWKNATIPLFCAT VTVFYGVPTWKNAT CIQYVTVFYGVPAWRNATIPLFCAT VTVFYGVPAWRNAT Peptide according to claim 6, characterized in that it has one of the formulas: 20 CTQYVTVFYGVPTWKNATIPLFCAT VTVFYGVPTWKNAT CIQYVTVFYGVPAWRNATIPLFCAT VTVFYGVPAWRNAT Peptide according to claim 2, characterized in that it exhibits one of the general formulas: DK 174705 B1 X-E - L-NVTE-F - W-NZ XL-NVTE-FZ 5 wherein X and Z are OH or N 2 groups, or when the immunological properties of peptides liberated from these groups are not significantly modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which enable the indicated peptide to retains the immunological properties of one of the following sequences: DDYQEITL-NVTEAFDAWNN L-NVTE DDYSELAL-NVTESFDAWEN 15 PNPQEWLVNVTENFNMWKN LVNVTE Peptide according to claim 8, characterized in that it has one of the formulas: 20 DDYQEITL-NVTEAFDAWNN L-NVTEAF DDYSELAL-NVTESFDAWEN L-NVTESF 10 -GGDPEITTHSFNCRGEFFYCNTSKLFN NCRGEFFYCN -GGDPEITTHSFNCGGEFFYCNTSGLFN NCGGEFFYCN 10 YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT 10 NHCNTSVITESCD NTSVIT NHCNTSVIQECCD NTSVIQ TSCNTSVITQACP 15 NTSVIT Peptide according to claim 2, characterized in that it exhibits one of the general formulas: DK 174705 B1 XL-S-KPCVKL-PLC-Z XKPCVKLTPLCVZ XS-KPCVKLTPLCVZ 5 wherein X and Z are OH or NH immunological properties of peptides liberated for these groups are not substantially modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which allow the peptide to retain the immunological properties of one of the following sequences: LFETSIKPCVKLTPLCVAMK LFETSIKPCVKLSPLCITMR 15 LWDQSLKPCVKLTPLCVSLK KPCVKLTPLCV KPCVKLSPLCI SLKPCVKLTPLCV Peptide according to Claim 10, characterized in that it exhibits one of the following structures: Peptide according to claim 2, characterized by. that it contains the basic structure: X-NSI-CZ DK 174705 B1 XN-SIZ wherein X and Z are OH or NH 2 groups, or when the immunological properties of peptides released for these groups are not significantly modified, X and Z may represent groups comprising 1 -5 amino acid residues, and each of the hyphens denotes an aminoacyl residue selected from those which allow the indicated peptide to retain the immunological properties of one of the following sequences: Peptide according to claim 12, characterized in that it has one of the formulas: Peptide according to claim 2, characterized in that it has the general formula: ♦ I DK 174705 B1! XYC-PGALCN-TZ wherein X and Z are OH or NH 2 groups or when the immunological properties of peptides released for these groups are not substantially modified, X and Z may represent groups comprising 1-5 amino acid residues and each of the hyphens denotes an aminoacyl residue selected from those which enable the indicated peptide to retain the immunological properties of one of the following sequences: The peptide of claim 14, characterized in that it has one of the formulas; no: 15 YCAPPGYALLRC-NDT YCAPAGFAILKCNNKT YCAPAGFAILKCNDKK YCAPAGFAILKCRDKK 20 15 EKLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT Peptide according to claim 2, characterized in that it has the general formula: X ----- aC ---- WZ wherein X and Z are OH or NH 1 groups or when the immunological properties of peptides are liberated from these groups are not substantially modified, X and Z may represent groups comprising 1 -5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which allow the indicated peptide to retain the immunological properties of one of the following sequences: NKRPRQAWCWFKG-KWKD DK 174705 B1 NERPKQAWCRFGG-NWKE N-MRQAHCNISRAKWNA Peptide according to claim 16, characterized in that it has one of the formulas: NKRPRQAWCWFKG-KWKD NERPKQAWCRFGG-KWKE N-MRQAHCNISRAKWNA 10 D-IRRAYCTINETEWDK 1-IGQAHCNISRAQWSK Peptide according to claim 2, characterized in that it has one of the general formulas: XG-DPE --- NC-GEF-YC ---- NZ XNC-GEF-YC-Z I wherein X and Z are OH or NH 2 groups, or when the immunological properties of peptides liberated from these groups are not significantly modified, X and Z may represent groups comprising 1-5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those enabling the indicated peptide retains the immunological properties of any of the following sequences: 25 KGSDPEVAYMWTNCRGEFLYCNMTWFLN NCRGEFLYCN GG-DPEVTFMWTNCRGEFLYCKMNWFLN NCRGEFLYCK 30 -GGDPEIVTHSFNCGGEFFYCNSTQLFN NCGGEFFYCN DK 174705 B1 A peptide according to claim 18, characterized in that it exhibits one of the following structures: Peptide according to claim 2, characterized in that it exhibits one of the general formulas: X-ClQ1 - G-YZ XC-IQ-IZ wherein X and Z are OH or NH2 groups or when the immunological properties of peptides liberated for these groups are not substantially modified, X and Z may represent groups comprising 1 -5 amino acid residues, and each of the hyphens represents an aminoacyl residue selected from those which allow the indicated peptide to retain the immunological properties of one of the the following sequences: RNYAPCHIKQIINTWHKVGRNVY CHIKQII 30 RNYVPCHIRQIINTWHKVGKNVY CHIRQII TITLPCRIKQFINMWQEVGKAMY CRIKQFI! DK 174705 B1 20 NHCNTSVITESCD NTSVIT NHCNTSVIQECCD NTSVIQ TSCNTSVITQACP 25 NTSVIT INCNTSVITQACP NTSVIT INCNTSAITQACP NTSAIT 30 Peptide according to claim 20, characterized in that it exhibits one of the structures: 22. Peptide exhibiting peptide structure common to envelope glycoprotein pep * time skeleton from HIV-2, characterized in that it also exhibits a peptide structure common to the peptide skeleton of the ENV amino acid sequence of SIV-1 shown in Figure 3. 20 Antigenic agent containing the peptide env according to claim 22 or at least one peptide according to claims 3, 4 and 5 or at least one oligomer of such peptide, characterized in that it specifically recognizes the presence of antibodies against HIV-2. 25 An immunogenic agent containing all or part of the peptide env according to claim 22 or at least one peptide or at least one oligomer of this peptide or peptide according to claims 6-21 conjugated with a carrier molecule, in combination with a pharmaceutically acceptable carrier for the preparation of vaccines , characterized in that it induces the formation of antibodies against these peptides in sufficient quantity to effectively inhibit proteins from HIV-2 retroviruses, or even HIV-2 retroviruses themselves. DK 174705 B1 Immunogenic agent according to claim 24, characterized in that it contains peptides having sequences which exhibit an amino acid homology of over 50% in envelope glycoproteins of HIV-2, SIV-1 and HIV-1. 25 PNPQEWLVNVTENFNMWKN LVNVTENF PNPQEIELENVTEGFNMWKN LENVTEGF PNPQEIALENVTENFNMWKN 30 LENVTENF 25 EKLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT EDLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT DN LWVTVYY GVPVWKEATTTLF CAS 30 VTVYYGVPVWKEAT An immunogenic agent according to claim 24 or 25, characterized in that it contains at least one peptide or at least one oligomer of such peptide or peptide conjugated with a carrier molecule, which peptide is selected from env4, env5. env6 and env10. A method for in vitro diagnosis of HIV-2 infections in a biological fluid, characterized in that the biological fluid is contacted with at least one peptide according to any one of claims 1-5 or a conjugate of these peptides with a carrier molecule, or in contact with the peptide env according to claim 22, and detects the possible presence of an antigen-antibody complex in the biological fluid by physical or chemical means. A method for in vitro diagnosis of HIV-2 infections in a biological fluid according to claim 27, characterized in that the detection of the possibly formed antigen-antibody complex is carried out by immunoenzymatic tests (type ELISA), immunofluorescence tests (of IFA type), radioimmunological specimens (RIA type) or radioimmunoprecipitation samples (RIPA type). A kit for in vitro diagnosis of HIV-2 infections in a biological fluid, characterized in that it comprises: a peptidic agent containing a peptide according to any one of claims 1-5, or a mixture of such peptides or a conjugate of these peptides 30 with a carrier molecule, or the peptide env according to claim 22, a reagent for composition of a medium favorable for carrying out an immunological reaction, one or more optionally labeled reagents for detecting the antigen-antibody complex formed by the immunological reaction and a biological reference fluid free of antibodies which is recognized by said peptidic acid! in