DK176372B1 - NEw peptide(s) with immunological properties of HIV-2 envelope protein - have the structure of simian immune deficiency virus proteins, useful in diagnosis and of vaccine components - Google Patents

Abstract

New peptides (I) have immunological properties in common with those of the peptide skeleton of the envelope protein of HIV-2 and also have a peptide structure in common with that of SIV (simian immunodeficiency virus)-7 glycoprotein. Also new are (i) a sequence of 9600 nucleotides corresponding to the SIV genome (reproduced in the specification, together with derived amino acid sequence of viral proteins gag, pol, env, Q, X, R, tat, art and F genes) and its fragments; (2) recombinant DNA contg. all or part of the CDNA from this sequence inserted into a vector, and (3) antigenic and immunogenic conjugates contg. (I).

Description

DK 176372 B1

The present invention relates to peptides having immunological properties, possibly immunogenic properties, which are common to purified antigens obtainable from viruses capable of provoking lymphadenopathies which can develop into acquired immunodeficiency syndrome 5 (AIDS) in humans.

The present invention relates, in a first aspect, to an antigenic peptide Gaol, characterized in that it exhibits one of the basic structures:

10 XDCKLVLKGLGMNPTLEEMLTAZ XDCKLVLKGLGTNPTLEEMLTAZ

wherein X and Z are OH or NH 2 groups, or when the immunological properties of peptides 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 such groups. which allows the specified peptide to retain the immunological properties of one of the following sequences:

20 DCKLVLKGLGMNPTLEEMLTA DCKLVLKGLGTNPTLEEMLTA

In another aspect, the invention relates to an antigenic agent containing at least one peptide gaol of the invention or at least one oligomer thereof, which antigenic agent is characterized in that it is capable of being recognized by biological fluids of human origin, in particular serum containing HIV-2 antibodies and to some extent anti-HIV-1 antibodies.

In a third aspect, the invention relates to a method for in vitro diagnosing HIV-2 infections in a biological fluid, which method is characterized by contacting the biological fluid with at least one peptide of the invention or a conjugate thereof. peptides with a carrier molecule, and detects the possible presence of an anti-antibody complex in the biological fluid by physical or chemical means.

In a fourth aspect, the present invention relates to in vitro diagnostic kits for HIV-2 infections in a biological fluid, which kits are characterized by comprising: a peptidic agent containing a peptide of the invention or a mixture of such peptides or a conjugate of these peptides with a carrier molecule, or, 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 antibody-free biological reference fluid which is recognized by said peptidic agent.

20

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. 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 868-25 871.

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

30 3 DK 176372 B1 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.

5

They have a reverse transcriptase activity which necessitates the presence of Mg2 + 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.

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 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.

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 numerous African disease cases where symptoms of lymphadenopathy or AlDS occur.

HIV-2 retroviruses are distinguished, as are HIV-1 retroviruses, 30 by a tropism against human T4 lymphocytes and by a cytopathogenic effect against 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 5 T4 lymphocytes) and immortalized cells derived from these T4 cells. 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-10 oligodeoxythymidylase) (poly (A) -ol igo (dT) 12-18) ; 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 15 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 that is not immunologically recognized by the protein p19 of HTLV-I or HTLV-II virus in radioimmunoprecipitation assay; They further 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 cross immunologically with the envelope glycoprotein gp140 from STLV-III (virus isolated from monkeys); These lysates further contain antigens which can be labeled with 35S cysteine, whose molecular weight is between 32,000 and 42,000-45,000: they further comprise an antigen having a molecular weight of the order of 36,000 and an antigen having a molecular weight of the order of 42,000, one of these antigens (referred to as p36 and p42) likely being a transmembrane glycoprotein of HIV-2 virus, the genomic RNA of HIV-2 does not hybridize with genomic RNA of HIV-10 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; 15 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-III, 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.

A strain of retrovirus SIV-1 mac 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 structural proteins or glycoproteins obtainable under analogous conditions from HIV-2. This retrovirus SIV-1, whose infectious nature is 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).

10

Using the same techniques as described above, it has been found that one can also obtain from SIV-1 mac: a basic 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, 20 which 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-25 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, 1986 by CNCM under I-522 under the reference designation LAV-II 30 ROD.

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

7 Furthermore, further studies of other retroviruses have also made possible a mapping of their complete nucleotide sequences. This is especially true for cDNA derived from genomic RNA from SIV virus.

5

Cloning and sequencing of SIV-1 mac virus enabled mapping of its nucleotide sequence and was performed under the following conditions: 10 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 into the BamHI restriction site of the bacteriophage λ vector ELBL3 to obtain a genomic library. The 2 million recombinant phages in the thus compiled genomic library were examined in situ under security 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 25 right-sided LTR is complete.

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

Thirty 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). 1.2).

8 DK 176372 B1

The results are given in the figures described below.

FIG. 1B denotes the nucleotide sequence of the genome from the 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. 1C shows comparison of the theoretical products of the viral genes and of LTR between HIV-2 and SIVmac (ASIV-1).

10

The nucleic acid sequences of cIV DNA 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.

1 C). This presentation as recognized from the complete genome by comparing FIG. 1B with FIG. Figures 3-11 allow for finding or deducing the nucleic acids having essential structural elements common to the two viruses.

The obtained cDNAs from SIV or their fragments (or recombinants containing them) can be used as probes to diagnose the possible presence of HIV-2 virus in samples of serum or other fluids or biological tissues from patients suspected of being carriers. of HIV-2 virus. These probes are also preferably labeled (with radioactive, enzymatic or fluorescent or similar markers). Such interesting probes for the diagnosis of HIV-2 virus or a variant of HIV-2 can be characterized in that they consist of the complete cDNA or a fraction thereof from the genome of SIV virus or in particular recombinant fragments contained in various clones.

The probes that can be used to diagnose HIV-2 virus and in diagnostic kits are by no means limited to the probes described above. On the contrary, they encompass all the nucleotide sequences derived from the genome of SIV virus, a variant of SIV or a structurally related virus, as long as they allow the determination of antibodies directed against HIV-2 or a related virus in biological fluids from people suspected of developing AIDS.

r

The detection can be carried out in any manner known per se. It can be effected by contacting the probes either with nucleic acids obtained from cells contained in these sera or other biological media, e.g. cephalospinal fluids, saliva, etc. It may also be effected by contacting these probes with the media itself once their nucleic acids are made available for hybridization with the probes, and this under conditions which permit hybridization between the probes and the nucleic acids. The final diagnostic step in vitro then involves the detection of the possibly induced hybridization. This diagnosis, which uses hybridization reactions, can also be carried out using mixtures of probes originating from a HiV-2 and a SIV-1 virus or an HIV-1, HIV-2 and SIV, respectively. it is necessary to make a difference between the types of virus sought.

In general, the method for diagnosing the possible presence of HIV-2 virus or a variant thereof in serum or other fluids or tissue samples obtained from patients suspected of carrying HIV virus includes the following steps: 1) at least one hybridization steps performed under stringent conditions by contacting DNA from cell samples of the suspected patient with one of the above labeled probes on a suitable membrane; 2) washing the membrane with a solution ensuring the preservation of the stringent hybridization conditions; 3) detecting the possible presence of HIV-2 virus by an immunodetection method.

10 DK 176372 B1

The hybridization can be carried out under non-stringent conditions and the leaching of the membrane takes place under conditions adapted to the hybridization conditions. Nucleic acids corresponding to the sequences located in regions analogous to variants of SIV as well as to all nucleic acids whose modifications are due to the degeneration of the genetic code may also be used.

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 oroteins. is also described in the above EP application No. 87 / 400,151.4. These results show that core proteins (gag proteins) in HIV-2 exhibit less distinct differences from HIV-1 virus cells than the envelope proteins (env proteins). Overall, the env proteins in HIV-2 have been shown to exhibit extremely weak, if nonexistent, immunological similarities to the corresponding env proteins in the HIV-1 virus.

In contrast, comparative studies between the structures of the cDNA sequences in HIV-2 and SIV virus have enabled the identification of certain 20 common characteristics that appear at the protein level.

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

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

These findings are not only apparent in terms of molecular weights: 130-30 140 kd for the major HIV-2 and SIV glycoproteins versus ca. 110 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-1mac, whereas the same serum samples and the same HIV-2 antibodies in similar experiments do not recognize gp110 from 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 5 HIV-2 extracts.

The major core protein of 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.

10

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.

15

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 structures of the gag and env proteins for HIV-2 or from SIV, and even from HIV-1. These peptides are particularly useful for diagnosing an infection in people with HIV-2 virus or a variant thereof.

Against this background, methods and means for in vitro diagnosis of antibodies against HIV-2 viruses or variants thereof can be developed, especially in 25 biological samples, in particular serum samples from patients who have been infected with HIV-2 virus, some of these 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 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 suitable for the preparation of active ingredients of vaccines against HIV virus, and thus against AIDS.

In order 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 of the following table: M Methionine L Leucine I Isoleucine 15 V Vaiin F Phenylalanine S Serine P Proline T Threonine 20 A Alanine Y Tyrosine H Histidine Q Glutamine N Asparagine 25 K Lysine D Aspartic Acid E Glutamic Acid C Cysteine W Tryptophan 30 R Arginine G Glycine 13 DK 176372 B1 When an amino acid due to its position in the middle of the amino acid chain characteristic of a given peptide may have several meanings, it may either be indicated by a hyphen if its meaning may be arbitrary, or by a lower case when this amino acid may exhibit a number of forms. drawn meanings, however, and this number is always greater than 1. In the latter case, the possible meanings of the lowercase letter are always indicated in connection with the peptide to which it belongs.

For ease of reading, these peptides are also designated with a shortened env or gag followed by a number, by reference to the contained amino acids as appropriate, either in the env proteins or in the gag proteins of certain types of HIV-1, HIV 2 or SIV, This is discussed in more detail below.

In the following definitions, the groups X denote either a free NH 2 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 earlier, and groups 2 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 whose 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 the conservation in essentially the immunological, possibly immunogenic, properties of the peptides from which they are ironed.

Peptides which have immunological properties in common with antigens from 30 HIV-2 and for some of them further with antigens from HIV-1 or variants thereof have a peptide structure in common with antigens from SIV. Advantageously, these peptides usually comprise a maximum of 40 amino acid residues.

14 DK 176372 B1

Examples of such peptides are the following: env1

XRV-AIEKYL-DQA-LN-WGCAFRQVCZ

5 env2

X-LE-AQI-QQEKNMYELQKLNZ

Env3

10 XELGDYKLVEITPIG-APT-KR ----- Z

env4 X — VTV-YGVP-WK-AT - LFCA-Z 15 env5 X — QE-L-NVTE-F-W-NZ env6

XL-S-Z-KPCVKLTPLCV

20 env7 X — N-S-IT - C-K — Z env8 25 X-1 — YC-P-G-A-L-C-N-TZ env9

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

30 env10

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

env11 15 DK 176372 B1

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

And more specifically: 5 env1

XRV-AIEKYL-DQA-LN-WGCAFRQVCZ

env2

X-LE-AQIQQEKNMYELQKLNZ

10 env3

XELGDYKLVEITPIG-APT-KR Z

env4 15 X — VTV-YGVP-W-AT-LFCA-Z env5 X — E — L-NVTE-F — W-NZ 20 env6 XL — S-KPCVKL-PLC — Z env7 X — NS1 — CK — Z 25 env8 X1 — YC-PGALCN-TZ env9

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

env10

Z-G ------ DPE-NC-GEF-YC ------ NZ

16 DK 176372 B1 env11

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

Particularly advantageous peptides similar to the above exhibit the following formulas: env1

XRVTAIEKYLQDQARLNSWGCAFRQVCZ, or XRVTAI EKYLKDQAQLNAWGCAFRQVCZ

env2 XSLEQAQIQQEKNMYELQKLNSWZ, or XLLEEAQIQQEKNMYELQKLNSWZ 15 env3

XELGDYKLVEITPIGFAPTKEKSSSSAHZ, or XELGDYKLVEITPIGLAPTNVKRYTTG-Z

It is noted that the peptides env1, env2, env3 confirm the very high 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.

env4 25 XabsdVTVeYGVPfWogAThiLFCAjZ, in which the letters a-j and o can have the following meanings:

a is C, E or D 30 b is T, K, D, N or I c is Q or L d is Y or W e is F or Y

17 DK 176372 B1

f is T, V or A g is N or E h is I or T i is P or T 5 j is T or S

0 is K or R

env5

XabcoEdeLfNVTEgFhiWjNZ, 10 in which the letters a-j and o can have the following meanings:

a is D or P b is D or N 15 c is Y or P d is I, V, I or L e is T, V, E or A f isV, G or E or -g is A, N, G or S 20 h is D or N

1 is A or M

j is N, K or E o is Q or S

25 env6 XLabcSdKPCVKLoPLCuefKZ, in which the letters a-f, o and u can have the following meanings:

30 a is F or W b is E or D c is T or Q d is I or L

18 DK 176372 B1 e is A (S or T f is M or L o is T or S u is V or I 5 env7

XabCNxSylocdCeKfghiZ, wherein the letters a-i and x and y can have the following meanings: 10 a is N or T or I b is H or S or N c is E or G d is S, A or C 15

e is D or P f is H, or D g is Y or S h is W or F 20 is D or E X is T or R y is V or A o is T or Q

25 env8

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

30 a is A or P b is R or P c is F, I or C d is R or H

19 DK 176372 B1

e is P or A f is Y or F g is L or I h is R or K 5 i is-or N j is D or K x is A or T

env9 10 Xwa bcxyAd Cefg h izWjkZ, in which the letters a-k and x-z can have the following meanings: a is K or - or E 15 b is R or-

c is P or M or I d is W or H or Y e is W or N or T or R f is F or I

20 g is K or S or N or G 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 25 w is N, D or I

x is R or G or K y is Q or K or R z is K or E or Q or N

30 env10

XaGbDPEcdefghNCiGEFjYCokxImnNZ, in which the letters a-n and x can have the following meanings: 20 DK 176372 B1

a is K or - or G b is S or G or -c is V or I

5 d is A or Veller T

e is Y or T or M or F f is M or H g is W or S h is T or F 10 i is R or G j is L or F

0 is N or K k is M or S

15 I is W or Q or Keller G m is F or L n is L or F x is T or S or N

20 env11

Xa bed wCe I oQfl xg yh izGj kl YZ, in which the letters a-l and w-z can have the following meanings:

25 a is R or T or S or N b is N or I c is Y or T d is A or L or V e is H or R 30 is I or F g is T or M h is H or Q or A

1 is K or E

21 DK 176372 B1

j is R or K k is N or A I is V or M w is P or Q 5 x is N or K y is W or V z is V or T or K o is K or R

The structure of the antigenic peptide for which the gene is encoded and designated qaq1 is represented by: XDCKLVLKGLGaNPTLEEMLTAZ, wherein a represents M or T.

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 for peptides listed above, appear to correspond to identical amino acids located in the same order in the corresponding env or gag sequences of the env or gag proteins of at least one HIV or SIV-1 virus.

The positions of these sequences are underlined and distributed within the amino acid sequences of the env 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.

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

The unbroken lines shown at certain locations 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 two 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.

Peptides may also be modified by insertion and / or deletion 5 and / or substitution of one or more amino acids, as long as the antigenic or immunogenic properties of these peptides are not modified, or the recognition properties of the antigen or antibody of these peptides are not substantially modified.

Examples of peptides having immunological properties in common with the peptide backbone of the envelope glycoprotein of virus of class HIV-2, these peptides containing a number of amino acid residues not exceeding 40 are as follows: 15 env1

RVTAIEKYLQDQARLNSWGCAFRQVC

AIEKYLQDQ

RVSAIEKYLKDQAQLNAWGCAFRQVC

AIEKYLKDQ

20 env2

SLEQAQIQQEKNMYELQKLNSW

QIQQEKN

LLEEAQIQQEKNMYELQKLNSW

25 env3

ELGDYKLVEITPIGFAPTKEKSSSSAH YKLVEITPIGFAPTKKK ELGDYKLVEITPIGLAPTNVKRYTG-30 YKLVEITPIGLAPTNVK

env4

CTQYVTVFYG VPTWKNATIPLFCAT

23 DK 176372 B1

VTVFYGVPTWKNAT CIQWTVFYGVPAWRNATIPLFCAT VTVFYGVPAWRNAT EKLWVTVYYGVPVWKEATTTLFCAS 5 VTVYYGVPVWKEAT

EDLWVTVYYGVPVWKEATTTLFCAS VTVYYGVPVWKEAT DN LWVTVYYG VPVWKEATTTLFCAS VTVYYGVPVWKEAT

10 env5

DDYQEITL-NVTEAFDAWNN

L-NVTEAF

DDYSELAL-NVTESFDAWEN 15 L-NVTESF

PNPQEWLVNVTENFNMWKN

LVNVTENF

PNPQEIELENVTEGFNMWKN

LENVTEGF

20 PNPQEIALENVTENFNMWKN LENVTENF

env6

ETSIKPCVKLTPLCVAMK 25 ETSIKPCVKLSPLCITMR DQSLKPCVKLTPLCVSLK DQSLKPCVKLTPLCVTLN PCVKLTPLCV

30 env7

NHCNTSVITESCD

NTSVIT

NHCNTSVIQECCD

24 DK 176372 B1

NTSVIQ

TSCNTSVITQACP

NTSVIT

INCNTSVITQACP 5 NTSVIT

INCNTSAITQACP

NTSAIT

env8

10 YCAPPGYALLRC-NDT YCAPAG FAILKCNNKT YCAPAG FAILKCN DKK YCAPAG FAI LKCRDKK

15 env9

NKRPRQAWCWFKG-KWKD NERPKQAWCRFGG-NWKE N-MRQAHCNISRAKWNA D-IRRAYCTINETEWDK 20 l-IGQAHCNISRAQWSK

env10

KGSDPEVAYMWTNCRGEFLYCNMTWFLN

NCRGEFLYCN

25 GG-DPEVTFMWTNCRGEFLYCKMNWFLN NCRGEFLYCK

-GGDPEIVTHSFNCGGEFFYCNSTQLFN

NCGGEFFYCN

-GGDPEITTHSFNCRGEFFYCNTSKLFN 30 NCRGEFFYCN

-GGDPEITTHSFNCGGEFFYCNTSGLFN

NCGGEFFYCN

DK 176372 B1 env11

RNYAPCHIKQIINTWHKVGRNVY

CHIKQII

RN WPCHIRQIINTWHKVG KN W 5 CHIRQII

TITLPCRIKQFINMWQEVGKAMY

CRIKQFI

SITLPCRIKQIINMWQKTCKAMY 10 CRIKQII

NITLQCRIKQIIKMVAGR-Kaiy

CRIKQII

Gagi

15 DCKLVLKGLGTNPTLEEMLTA

Peptides can be advantageously 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 aminoacyl groups are successively condensed in the required sequence or condensed aminoacyl groups and pre-formed fragments which already contain several aminoacyl groups in the correct order, or more fragments thus prepared, it being understood that prior consideration has been given to protecting all the reactive groups on these aminoacyl groups or fragments, except for 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 methods well known in the art of 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 groups may be protected, e.g. with t-butyl ester groups.

For progressive synthesizing amino acid after amino acid, the synthesis preferably begins with a condensation of the C-terminal amino acid with the 10 amino acid corresponding to the adjacent aminoacyl in the desired sequence and then still up to the N-terminal amino acid. The method described by R.D. Merrifield, "Solid phase peptide synthesis" (J. Am. Soc., 45, 2149-2154) may also be used.

To prepare a peptide chain according to the Merrifield method, a very porous polymer resin is applied to which the first C-terminal amino acid is fixed in the chain. This amino acid is fixed to the resin by its carboxyl group while its amino group is protected, e.g. 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 protecting group for the amino group is t-butyloxycarbonyl, it can be removed by treating the resin with trifluoroacetic acid.

The second amino acid supplying 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.

27 DK 176372 B1

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.

10

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 portion of the peptide chain which is bound to the resin.

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

Monomeric peptides may also act as water-soluble oligomers of the above-mentioned monomeric peptides. The oligomerization can induce an increase in the immunogenicity of monomeric peptides. These oligomers may e.g. contain 2-10 monomer units.

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.

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

28 DK 176372 B1

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 and comprising the appropriate corresponding nucleotide sequences can be used.

15

Nucleic acids may contain one or more sequences derived from the cDNA sequence of HIV-2 ROD virus. These sequences, which are marked with the numbering that appear on the sequence described above, encode certain interesting peptides:

Sequence encoding env1 nucleotide 7850 - 7927 - - env2 - 8030-8095 - - env3 - 7601 - 7636 - - env4 - 6170-6247 25 - ... env5 - 6294-6349 - - env6 - 6392-6445 - - env7 - 6724-6763 - - env8 - 6794-6838 - - env9 - 7112-7162 30 - ... enviQ - 7253-7336 - - env11 - 7358-7426 - - gagl - 1535-1597 29 DK 176372 B1

Nucleotide sequences encoding the peptides env1-env11 and qaq1 can be located in FIG. 3 by comparison with the corresponding sequences described for HIV-2.

5 The nucleic acids corresponding to sequences located in analogous regions of the cDNA derived from variants of HIV-2 ROD or SIV, as well as all the nucleic acids whose modifications to the foregoing may have different genetic codes due to degeneration of the genetic code.

Conjugates obtained by covalently coupling peptides (or the aforementioned oligomers) to natural or synthetic physiologically acceptable and nontoxic carrier molecules by complementary reactive groups supported by the carrier molecule and peptide, respectively, can also be prepared. Examples of suitable groups are illustrated below:

Examples of carrier molecules or macromolecular carriers that may be included in the constitution of conjugates include natural proteins such as tetanus anatoxin, ovalbumin, serum albumin, hemocyamines, etc.

20

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, 25 and these generally exhibit a molecular weight in excess of 20,000.

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

In practice, it is advantageous to use 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 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-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.

Peptides can also be used in diagnostic methods to detect an infection with HIV-2 virus.

20

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.

25

A first group (I) comprises qaq1 peptides. These peptides recognize anti-HIV HIV-2 antibodies and are thus capable of detecting an infection with HIV-2. They also recognize to some extent anti-HIV-1 antibodies.

Another group (II) comprises peptides which correspond more specifically 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 transient or present infections due to the presence of HIV-2 antibodies. HIV in a person, more specifically a discrimination between infections provoked by HIV-2 and infections provoked by HIV-1.

5

Agents may be prepared containing at least one of the above peptides or at least one oligomer of such peptide which has the ability to be recognized by serum of human origin containing antibodies to HIV-2.

10

Methods for in vitro diagnosing one or more peptides to detect antibodies to HIV-2 in biological fluids, especially human serum, may include the following steps: The biological fluid is contacted with the peptides, the presence of a peptide-antibody complex detected by physical or chemical methods in the biological fluid.

Detection of the antigen-antibody complex can be performed by immunoenzymatic tests (ELISA type), immunofluorescence methods (IFA type), radioimmunological methods (RIA type) or radioimmunoprecipitation methods (RIPA type). Peptides can be labeled by a suitable marker, e.g. an enzymatic, fluorescent or radioactive marker.

Such methods may include the following steps: depositing determined amounts of a peptide-containing agent in a microtiter plate, placing increasing dilutions of the serum to be diagnosed in the wells, incubating the microtiter plate, and repeated rinsing thereof, introducing into the wells a labeled antibody to the immunoglobulins in the blood, 5 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 amount of substrate.

Kits for in vitro diagnosis of the presence of antibodies to HIV-2 virus and in some cases HIV-1 virus in a biological medium may include: a peptidic agent as described above, reagents to provide a composition of the medium suitable for carrying out the immunological reaction, 20 reagents enabling the detection of the antigen-antibody complex formed by the immunological reaction. Such reagents may also be labeled or 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.

Furthermore, antibodies to peptides can be prepared.

It goes without saying that this production is not limited to polyclonal antibodies.

33 DK 176372 B1

It also applies 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 described herein, and cells from a appropriate myeloma cell line and which can be selected for its ability to produce monoclonal antibodies that recognize the peptide originally used for the immunization of the animal.

Immunogenic agents can be used to prepare vaccines whose active ingredient consists of at least one peptide or oligomer of such peptide or peptide conjugated with a carrier molecule, and induces the formation of antibodies against these peptides in sufficient quantity to inhibit HIV-2 retrovirus proteins, even the HIV-2 retrovirus itself, in association with a pharmaceutically acceptable carrier.

Specifically, the immunogenic agents for the preparation of vaccines may comprise at least one of the peptides referred to above env4, env5. env6, env7, env8. env9. env10, env11 or mixtures thereof. Of these 20 peptides which are useful in constituting active ingredients in vaccines, some exhibit a basic amino acid structure corresponding to regions of envelope glycoproteins exhibiting a high degree of preservation, not only in HIV-2 and in SIV, but also in HIV-1 virus. Such peptides are termed env4. and certain of the peptides env5. env6 and envIO.

25

The immunogenic peptides or fragments thereof which are useful as active ingredients in vaccines, among the formulas corresponding to the sequences as in the envelope glycoproteins of HIV-2, SIV and HIV-1, may exhibit an amino acid homology exceeding 50% , and belonging to the external portion of the envelope of viruses, which are free or almost free of deletions, and which contain cysteine residues which promote the stability of the bonds and the formation of "hairpins".

34 DK 176372 B1

Examples of such peptides are: env4

XVTV-YGVP-W-ATZ

5 env5

XL NVTE-FZ

env6

XKPCVKL-PLC-Z

env7

XN-S-I-Z

15 env10

XNC-GEF-YC-Z

env11

XC-I-Q-IZ

20

Pharmaceutical agents may comprise of injectable solutions, suspensions or liposomes. These solutions may be present as suspensions or liposomes in a sterile isotonic aqueous phase, preferably a physiological saline or glucose solution.

Such suspensions, solutions or liposomes may be administered by intradermal, intramuscular or subcutaneous injection or by co-administration.

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

30

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

DK 176372 B1

These doses are given by way of example only.

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 examples 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 10 are aligned under the conditions similar to those described above in connection 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 HIV-15 1 deposited at CNCM with numbers I-232, I-240, 1-241, I-550 and 1-551.

Peptides can also be defined by the following formulas wherein X, Z and the hyphens have the above meanings:

XRV-AIEKYL-DQA-LN-WGCAFRQVCZ XAIEKYL-DZ

X-LE-AQIQQEKNMYELOQKLNSWZ 25 XQIQQEKNZ

XELGDYKLVEITPIG-APT-KR ----- Z

XYKLVEITPIG-APT-KRZ

30 X — VTV-YGVP-W-AT-LFCA-Z XVTV-YGVP-W-ATZ

X-E - L-NVTE-F-W-NZ

36 DK 176372 B1

XL NVTE-FZ

XL-S-KPCVKL-PLC-Z XKPCVKL-PLC-Z 5 XS-KPCVKL-PLC-Z

X — N-S-1 — C-Z XN-S-I-Z

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

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

NKRPRQAWCWFKG-KWKD

15

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

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

20

Other examples of peptides are as follows: 1) envelope proteins and glycoproteins encoded by the env gene. and shown in FIG. 3, 25 2) the protein GAG shown in FIG. 4, 3) the protein POL shown in FIG. 5, 4) the protein Q shown in FIG. 6, 5) the protein R shown in FIG. 7, 6) the protein X shown in FIG. 8, 30) The protein F shown in FIG. 9, 8) the protein TAT shown in FIG. 10th

37 DK 176372 B1

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 vertical points that appear between the two sequences correspond to amino acids common to the proteins of the two viruses.

5

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

1B.

These cDNA sequences located at the numbering of the 10 previously described sequences (Figure 1B) are as follows: - sequence encoding GAG nucleotides 551-2068. P0L 1726-4893 "" "Q," 4826-5467 15 - ...... "X," 5298-5633 ........ R, n 5637-5939 ........ F, "8569-9354 ....... .TAT-1, "5788-6084 ........ ART-1," 6014-6130 20 - ........ TAT-2, "8296-8391 ......" ART-2, "8294-8548" ENV, "6090-8732

The foregoing proteins may be used to diagnose the possible presence of antibodies directed against HIV-2 proteins, or even to the entire HIV-2, or for some of their use for the diagnosis of an infection due to an HIV virus. Thus, the peptide GAG encoded by the corresponding gene can be used to locate the possible presence of anti-HIV-1 or anti-HIV-2 antibodies. The ENV proteins 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.

38 DK 176372 B1

Thus, the present invention relates in a first aspect to an antigenic peptide aaq1. peculiar in that it exhibits one of the basic structures:

5 XDCKLVLKGLGMNPTLEEMLTAZ XDCKLVLKGLGTNPTLEEMLTAZ

wherein X and Z are OH or NH 2 groups, or when the immunological properties of peptides 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 such groups. which allows the specified peptide to retain the immunological properties of one of the following sequences:

15 DCKLVLKGLGMNPTLEEMLTA DCKLVLKGLGTNPTLEEMLTA

In another aspect, the invention relates to an antigenic agent containing at least one peptide qaq1 of the invention or at least one oligomer thereof, which antigenic agent is characterized in that it is capable of being recognized by biological fluids of human origin, especially serum containing HIV-2 antibodies and to some extent anti-HIV-1 antibodies.

The invention relates in a third aspect to a method for in vitro diagnosis of HIV-2 infections in a biological fluid, which method is characterized by contacting the biological fluid with at least one peptide according to the invention or a conjugate thereof. peptides with a carrier molecule, and detects the possible presence of an anti-gene antibody complex in the biological fluid by physical or chemical means.

In a preferred embodiment, the detection of the possibly formed antigen-antibody complex is performed by immunoenzymatic samples (type ELISA), immunofluorescence samples (type I FA), radioimmunological samples (type RIA) or radioimmunoprecipitation samples (type RIPA).

The invention relates in a fourth aspect to kits for in vitro diagnosis of HIV-2 infections in a biological fluid, which kits are characterized in that they comprise: a peptidic agent containing a peptide of the invention or a mixture of such peptides or a conjugate of these peptides with a carrier molecule, or, 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 which is free of antibodies recognized by said peptidic agent.

Claims (5)

1. Antigenic peptide qag1. characterized in that it exhibits one of the basic structures: 5 XDCKLVLKGLGMNPTLEEMLTAZ XDCKLVLKGLGTNPTLEEMLTAZ wherein X and Z are OH or NH 2 groups or when the immunological properties of peptides liberated for these groups are not substantially modified, X and Z may be denoted -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 any of the following sequences: 15 DCKLVLKGLGMNPTLEEMLTA DCKLVLKGLGTNPTLEEMLTA Antigenic agent containing at least one peptide qaq1 according to claim 1 or at least one oligomer thereof, characterized in that it is capable of being recognized by biological fluids of human origin, in particular serum containing anti-HIV-2 antibodies and in some extensive anti-HIV-1 antibodies. 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 claim 1 or a conjugate of these peptides with a carrier molecule, and detects the possible presence of an antigen-antibody complex in the biological fluid by physical or chemical means. 30 Method for in vitro diagnosis of HIV-2 infections in a biological fluid according to claim 3, characterized in that the detection of the possibly formed antigen-antibody complex is carried out by means of DK 176372 B1 immunoenzymatic samples (type ELISA), immunofluorescence samples (of IFA type), radioimmunological samples (RIA type) or radioimmunoprecipitation samples (RIPA type). Kit for in vitro diagnosis of HIV-2 infections in a biological fluid, comprising: a peptidic agent containing a peptide according to claim 1 or a mixture of such peptides or a conjugate of these peptides with a carrier molecule 10 coolant, or, 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 are recognized by said peptidic agent.