DK175879B1 - 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 175879 B1

The present invention relates to nucleotide sequences encoding peptides with immunological properties common to purified antigens obtainable from viruses capable of provoking lymphadenopathies which can develop into acquired immunodeficiency syndrome *, 5 ( AIDS) in humans. The invention further relates to recombinant nucleic acids comprising these nucleotide sequences.

More particularly, the invention relates to a nucleotide sequence comprising all or part of the nucleic acid sequence defined in FIG. 1B or 1C.

10

Antigenic peptides that can be recognized by antibodies induced in humans by virus are referred to as the abbreviation HIV (according to the nomenclature defined in NATURE). Peptides with immunogenic properties or peptides that can be immunogenic in vivo may manifest their immunogenicity by in vivo induction of antibodies that recognize the antigens characteristic of HIV-2 virus as well as (in some cases). these peptides) antigens characteristic of HIV-1.

These peptides can be used to prepare agents for the in vitro diagnosis of the potential incidence of certain forms of AIDS in humans and (for some of these forms) to prepare immunogenic agents and vaccines for use against HIV retroviruses.

Antibodies which can be induced in vivo by immunogenic peptides or peptides made immunogenic can be used to prepare active ingredients in drugs for human AIDS.

Some of the peptides can be used in methods for in vivo diagnosis of certain forms of AIDS in humans, and the peptides can also be used in 30 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

DK 175879 B1 I

No. 84 / 401,834 filed September 14, 1984. This virus is also present in I

have been described by F. Barre Sinoussi et al in Science 220. 45-99, pages 868-1

871. I

5 Variants of this HIV-1 virus, designated LAV, ELI and LAV MAL, are% I

has also been isolated, characterized and described in EP patent application I

No. 84 / 401,834. IN

The HIV-1 virus and its variants have the following properties:

10 I

As preferred target cells, the human Leu3 cells (or T4 lymphocytes) have I

and their "immortalized" cell derivatives. IN

They have a reverse transcriptase activity which necessitates presence

Of the Mg2 + ions, and exhibit a strong activity against poly (adenylate oligodeoxydymidylase) poly (A) oligo (dT) 12-18).

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

They have an average diameter of 139 nm and an average core diameter I

at 41 nm.

The lysates of these viruses contain a protein called p25 (chem. Protein), I

which does not exhibit immunological cross-linking with the protein p24 from HTLV-1.

25 I

They contain a protein called p42, which belongs to their sheath, and I

they additionally contain a envelope glycoprotein called gp110 which has an I

molecular weight of 110 000. I

30 The isolation and characterization of retroviruses belonging to a particular I

class and which has only a reduced immunological relationship with its I

origin, is disclosed in EP Patent Application No. 87 / 400,151.4. These retrovi- I

intoxication, which has been grouped under the designation HIV-2, has been isolated in I

3 DK 175879 B1 is associated with numerous African disease cases in which symptoms of a lymphadenopathy or an ALDS occur.

HIV-2 retroviruses are distinguished, as are HIV-1, 5 retroviruses, in 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.

Specifically, 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 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) (poly (A) oligo (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 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; Thirty lysates of these viruses contain a protein called p26 which does not exhibit immunological cross-over with protein p24 from HTLV-I virus or HTLV-II virus;

DK 175879 B1 I

these lysates also contain a protein called p16 which does not recover

is immunologically known by the protein p19 of HTLV-I or HTLV-II virus in ra-I

dioimmunopræcipitationsassay; IN

In addition, they contain a molecular weight envelope glycoprotein of size I

the order of 130,000-140,000 which does not exhibit immunological cross- I

gp110 from HIV-1, but which in turn cross immunologically I

with the envelope glycoprotein gp140 from STLV-III (virus isolated from monkeys);

These lysates further contain antigens which can be labeled with 35S-I

cysteine, whose molecular weight is between 32,000 and 42,000-45,000: they about

furthermore, a molecular weight antigen of the order of 36,000 I

and an antigen having a molecular weight of the order of 42,000, one of I

these antigens (referred to as p36 and p42) probably constitute an I

15 transmembrane glycoprotein from HIV-2 virus, I

the genomic RNA of HIV-2 does not hybridize with HIV genomic RNA.

1 under stringent conditions; IN

20 under non-stringent conditions, the HIV-I genomic RNA hybridizes

2 neither with the env gene nor with the LTR adjacent to the gene in HIV-1;

or with sequences from the pol region of the HIV-1 genome; IN

under non-stringent conditions, it weakly hybridizes with nucleotide side I

25 sequences in the HIV-1 region. IN

Another retrovirus called SIV-1, which replaces the early I

The term STLV-III used is isolated from rhesus monkeys (M.D. Daniel I

et al. Science 228, 1201 (1985) N.L. Letwin et al., Science 230, 71 (1985) I

30 under the designation "STLV-11mac"). IN

A further retrovirus named "STLV-IIUgm" (or SIVagm) is isolated in

in wild green monkeys. However, unlike the viruses present in the rhesus monkey, the presence of "STLV-IIIagm" does not appear to cause AIDS-type diseases in African green monkeys.

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 structural proteins or glycoproteins obtainable under analogous conditions from HLV-2. This retrovirus SIV-1, whose infectious nature has been found in monkeys, was named STLVIII by the researchers who isolated it (see prior to the bibliographic references above).

For linguistic reasons, these viruses are hereinafter referred to only by the term 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, f eg MAC or mac for rhesus monkeys or AGM for African green monkeys (abbreviation for African Green Monkey).

in

Using the same techniques as described above, it has been found that one can also obtain from SIV-1mac: a principal nuclear protein called p27, having a molecular weight of the order of 27 kd, 25 a larger envelope glycoprotein, called gp140, a protein called p32, which is probably a transmembrane protein that is rarely observed by radioimmunoprecipitation assay (RIPA) when the virus is previously labeled with 35S-cysteine, but which can be observed in 30 broad-band immunoassay samples.

More precise studies have been carried out on the above viruses HIV-2 and SIV viruses. Continued studies of HIV-2 retroviruses have also led

I DK 175879 B1 I

I I

In obtaining complementary DNA sequences (cDNA) for RNA in their region

In norns. The complete nucleotide sequence of cDNA for a representative I

In the HIV-2 (HIV-2-ROD) retrovirus is deposited on February 21st

In 1986 at CNCM with the number I-522 under the reference designation LAV-II I

I 5 ROD. IN

I This nucleotide sequence and the open reading frames that they contain are I

indicated in Figure 1 A. I

In 10 1, further studies of other retroviruses have also made it possible for a card I

In laying down their complete nucleotide sequences. This is especially true of you

for cDNA derived from genomic RNA from SIV virus. IN

In cloning and sequencing of SIV-1mac virus has enabled a mapping I

In addition to its nucleotide sequence and carried out under the following procedure

In things: I

In DNA from HUT 78 cells infected with SIV virus (isolate STLV-III mac 142-83 I)

As described by Daniel et al (1985) Science, 228 pages 1201-1204), and in part I

In digestion with the restriction enzyme Sau3A was cloned into BamHI-I

At the restriction site of the bacteriophage λ vector ELBL3 to obtain a gel I

In nomic library. The 2 million recombinant phages in the thus assembled

In situ genomic libraries were investigated in situ under safety assessment

In moods P3 using sequences of HIV-2 virus derived from clone I

In the lambda-ROD4, lambda-ROD35 and E2 fields (Clevel et al (1986) Nature, I

In 324. page 691) and was nick-translated. IN

In Hybridization, 5xSSC was performed at 50 ° C and the leaches I

I at 2xSSC at 50 ° C. A single clone containing all virus sequences

In 30 watches were obtained. This clone was named lambda-SIV-1. The insertion in phage I

In lambda-SIV-1 measures 16.5 kb in total and includes an integrated provirus where I

In it, only the first 250 bases are missing from the left-hand LTR, while the I

In the right-hand LTR is complete. IN

7 DK 175879 B1

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

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). 1.2).

10 The results are given in the figures described below.

FIG. 1B 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.

15

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).

The invention relates to cDNA fragments derived from cDNA derived from the complete SIV-1 genome, these fragments containing one or more sequences derived from the complete cDNA sequence, and encoding the interesting peptides of the invention and more particularly those sequences which is shown in FIG. 1B and in FIG. 1C as far as the LTR sequence in viruses is concerned.

25

The invention is described in the following with reference to the drawings, in which:

Figure 1A shows the complete nucleotide sequence of cDNA for a representative HIV-2 class retrovirus and the open reading frames that they contain.

Figure 1B shows the nucleotide sequence of the genome from SIV virus and the se I

sequences derived therefrom for the viral proteins corresponding to the products I

H of the genes gag, pol, env, Q, X, R, tat, species, F I

Figure 1C shows the nucleotide sequences of SIV cDNAs positioned against I

In the nucleotide sequences of the HIV-2 ROD virus as far as the LTR sequence I is concerned

Figure 2 shows the nucleotide sequences of HIV-2 cDNA placed against I

the nucleotide sequences of HIV-1 I

I 10

In Figure 3, comparison of the theoretical products of the env gene shows the I-1

In limb HIV-ROD and SIV-mac I Figure 4 shows comparison of the theoretical products of the gag gene between I 15 limb HIV-ROD and SIV-mac I Figure 5 shows comparison of the theoretical products of the pol gene between HIV-ROD and SIV-mac I 20 Figure 6 shows comparison of the theoretical products of the Q gene between I HIV-ROD and SIV-mac I Figure 7 shows comparison of the theoretical products of the R-gene between I HIV-ROD and SIV -mac I 25 I Figure 8 shows comparison of the theoretical products of the X gene between I HIV-ROD and SIV-mac

Figure 9 shows comparison of the theoretical products of the F gene between 30 HIV-ROD and SIV-mac

Figure 10 shows comparison of the theoretical products of the tat gene between HIV-ROD and SIV-mac DK 175879 B1 9

Figure 11 shows comparison of the theoretical products of the species gene between HIV-ROD and SIV-mac.

<5 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.

1C). 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 having essential structural elements common to the two viruses.

The obtained cDNA 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 of patients suspected of be 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 of the SIV virus genome or especially recombinant fragments contained in various clones.

The probes used in the diagnosis of 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 the SIV virus, a variant of the SIV or a structurally related virus, as long as they allow the determination of antibodies directed against HIV-2 or a related virus in human biological fluids. who are suspected of developing AIDS.

30

The detection can be carried out in any manner known per se. It may be effected by contacting the probes either with nucleic acids obtained from cells contained in these sera or other biological media.

I DK 175879 B1 I

I 10 I

In e.g. cephalospinal fluids, saliva, etc. It can also be done by:

These probes are brought into contact with the media itself, once they are

Nucleic acids are made available for hybridization with the probes, and this I

Under conditions which allow a hybridization between the probes and

In the 5 nucleic acids. The final diagnostic step in vitro then comprises detection I

In the ring of the possibly induced hybridization. This diagnosis that makes you

In the use of hybridization reactions, may also be carried out by means of I

mixtures of probes originating from an HIV-2 and a SIV-1I, respectively

In viruses or an HIV-1, HIV-2 and SIV when no need to do it

In 10 distinctions between the virus types sought. IN

In general, the method for diagnosing any present I - comprises

In the room of HIV-2 virus or a variant thereof in serum samples or other tissues

Do you have tissue or tissue samples obtained from patients suspected of being impaired?

In 15 viruses of HIV virus, the following steps:

At least one hybridization step carried out under stringent conditions;

You contact DNA of cell samples from the suspected patient

In one of the above labeled probes on a suitable membrane,

I 20 I

2) washing the membrane with a solution that ensures the preservation of the membrane

In stringent hybridization conditions, I

3) detecting the possible presence of HIV-2 virus by an im-I

In 25 mouth detection method. IN

In another preferred embodiment of the method according to the invention

In the process, said hybridization is carried out under non-stringent conditions

In conditions and the leaching of the membrane occurs under conditions that are I

In 30 adapted to the hybridization conditions. It goes without saying that the invention I

You also refer to the nucleic acids corresponding to the sequences located in I

In regions analogous to variants of SIV as well as all nucleic acids, if I

In modifications is due to the degeneration of the genetic code. IN

11 DK 175879 B1

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. is also described in the above-mentioned 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 found to exhibit extremely weak, if nonexistent, immunological similarities to the corresponding env-10 proteins in the HIV-1 virus.

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.

15

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 25 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 in particular 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 30 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.

I DK 175879 B1 I

I 12 I

In The major chemical protein from HIV-2 appears to exhibit a mediol. · I

cooling weight of approx. 26,000, which is between the molecular weight of p25 from HIV · I

In 1 and p27 from SIV. IN

5 These observations stem from experiments conducted on virus extracts I

I obtained from HIV-2 isolated from one of the above patients. Similar I

In results obtained with virus extracts of HIV-2 isolated from another pa- I

In ten. IN

In 10 more extensive experiments, the inventors recognized a first class I

I of peptides having amino acid sequences that are either identical or nearly I

In related to sequences contained within the interior of the structures of gag- I

and the env proteins for HIV-2 or from SIV, and even from HIV-1. These pep- I

times are particularly useful for diagnosing an infection in humans

15 with HIV-2 virus or a variant thereof. IN

These extensive studies have also led to the possibility of synthesizing I

peptides that are immunogenic or can be immunogenic and which are shown

You see structural characteristics that enable them to induce in vivo

In the provision of antibodies capable of recognizing env proteins both from I

In HIV-1 and HIV-2, and at least for some of these peptides

In order to bind both to HIV-1 virus and to HIV-2 virus, especially with the I

In order to neutralize these. The use of these types of peptides is thus I

In particular, it is indicated for the preparation of active ingredients of vaccines against I

In 25 HIV viruses, and thus against AIDS. IN

In order to describe below the amino acid residues included in the structure of the I

In the present peptides, the unique interna I is used for the amino acid

In tional nomenclature, denoting each natural amino acid with a large I

In 30 letters according to the following table:

I M Methionine I

I L Leucine I

---------------------- -; - DK 175879 B1 13 I Isoleucine V Valin F Phenylalanine S Serine 5 P Prolin T Threonine A Alanine Y Tyrosine 10 H Histidine Q Glutamine N Asparagine K Lysine D Aspartic Acid 15 E Glutamic Acid C Cysteine W Tryptophan R Arginine G Glycine 20 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 that 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.

To facilitate reading, these peptides are also designated with an abbreviation 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.

I DK 175879 B1 I

I I

In general, it should be noted that the amino acids which have a unique meaning

(and is thus represented by a capital letter corresponding to it

international nomenclature), appears to correspond to identical ami- I

H acids located in the same order in the corresponding env- or - I

In 5 gag sequences in the env or gag proteins of at least one HIV or SIV-1 I

In viruses. IN

The positions of these sequences are underlined and distributed within amino acid I

In the sequences of the env proteins for HIV-2 ROD (CNCM No. I-I, respectively)

I-512) and HIV-1 BRU (CNCM No. I-232) shown in FIG. 2. The order of ami- I

In the nosymes of the env and gag proteins for SIV-1 mac, respectively (CNCM No. I

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

In the unbroken lines shown at certain locations of these sequences, un- I

It should be noted that certain amino acids contained in these sequences are optional

You loaded the presentation to allow identical amino acids to be brought

I in line (marked with an asterisk), or two vertical points on one and I

In the same vertical line in the corresponding protein sequences from on the I

On the one hand HIV-1 and HIV-2 and on the other hand SIV and HIV-2. IN

I 20 I

The peptides can advantageously be prepared by the classical methods of I

In peptide synthesis. This synthesis can be carried out in homogeneous solution or I

In clay in solid phase. IN

For example, in For example, the synthetic technique in homogeneous solution described in I can be used

I by HOUBEN-WEYL in "Method of Organic Chemistry", Ed. E. Wunsch, I.

In Volumes 15-1 and II., THIEME, Stuttgart 1974. I

I This method successively condenses two and two successive I

In 30 aminoacyl groups in the required order or condensed ami I

In noacyl groups and pre-formed fragments already containing several * I

In aminoacyl groups in the correct order, or more thus earlier I

In manufactured fragments, it is understood that prior art has been taken

In order 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 groups may be 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 of the invention, the method described by R.D. Merrifield, "Solid phase peptide synthesis" (J. Am. Soc., 45, 2149-21.54).

To prepare a peptide chain by 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. 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.

I DK 175879 B1 I

I 16 I

The second amino acid which supplies the second aminoacyl in I is then coupled

In the desired sequence from the C-terminal aminoacyl, to the debat-I

In the amino group of the first C-terminal amino acid fixed to the resin. IN

Preferably, the carboxyl group of the second amino acid is activated, e.g. with I

In dicyclohexylcarbodiimide and the amino group is protected, e.g. with t- · I

In butyloxycarbonyl. IN

Thus, the first portion of the desired peptide chain comprising - I

For 10 ter two amino acids and whose terminal amino group is protected. As early- I

re-protects the amino group and you can then proceed with fixation

In the ring of the third aminoacyl under analogous conditions to those using I

was reversed by the addition of the second C-terminal amino acid. IN

Thus, one of the amino acids to be fixed is fixed one after the other

You constitute the peptide chain, to the amino group, each time before being de-I

In protected, to the already formed portion of the peptide chain which is bound to I

In the resin. IN

Once the entire desired peptide chain is formed, the protective I is removed

In the groups of the various amino acids that make up the peptide chain, and I

You release the peptide from the resin, e.g. using hydrofluoric acid. IN

In water-soluble oligomers of the above-mentioned monomeric peptides, you can

In 25, induce an increase in the immunogenicity of the monomeric peptides. IN

Without limiting this figure of speech, you cannot

Not to mention, however, that these oligomers e.g. may contain 2-10 mo- I

In number units. IN

The 30 monomer units included in this oligomer are either all of I

the polypeptide of sequence 1 or of the polypeptide of sequence 2 or of the I

one or the other of these polypeptides. IN

17 DK 175879 B1

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 containing the required number of monomeric units is obtained to obtain 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.

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.

The invention relates to nucleic acids containing one or more sequences derived from the cDNA sequence of HIV-2 ROD virus. These sequences, which are marked with 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 30 - --- env6 - 6392-6445 - - - env7 - 6724-6763 - - - env8 - 6794 - 6838 - - - env9-7112-7162

I DK 175879 B1 I

I 18 I

I .... envIO- 7253-7336 I

I .... env11- 7358-7426 I

I .... gaq1- 1535-1597 I

The invention also relates to the corresponding nucleic acids of SIV-I

In viruses containing one or more sequences derived from cDNA from SIV-1- I

In viruses. These sequences encoding the peptides env1-env11 and oaa1 can be I

In FIG. 3 by comparison with the corresponding se- I

In sequences described for HIV-2. IN

I 10 I

It goes without saying that the invention also relates to the nucleic acids corresponding to I

sequences located in analogous regions of cDNA derived from variants of HIV-2 I

In ROD or SIV, as well as all the nucleic acids, the modifications of which I

The foregoing is due to the degeneration of the genetic code. IN

I 15 I

In conjugates obtained by covalently coupling the peptides (or the aforementioned I)

oligomers) to natural or synthetic physiologically acceptable and non-I

In toxic carrier molecules, using complementary reactive groups, I

You are carried by the carrier molecule and the peptide, respectively. Examples of passport I

The 20 groups are illustrated below:

As examples of carrier molecules or macromolecular carriers which I

You may be included in the constitution of conjugates according to the invention, you may mention

In natural proteins such as tetanus anatoxin, ovalbumin, serum albumin

I 25, hemocyamines etc. I

As examples of synthetic carrier molecules, for example, mention poly- I

In lysines or poly (D-L-alanine) -poly (L-lysine). IN

In the literature, other types of useful macromolecular carriers are mentioned, I

I and these generally exhibit a molecular weight of over 20,000

19 DK 175879 B1

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,

Applied and Environmental Microbiology, (October 1981), Vol. 42, Nos. 4, 5,611-614 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 can be used where one or more reactive groups are used in the peptide and one or more reactive groups in the carrier molecules. 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.

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

As mentioned earlier, studies have made it possible to distinguish between two groups of peptides that can be used in methods for detecting 30 antibodies to HIV-2 virus in human biological fluids, especially serum or cephalospinal fluid.

I DK 175879 B1 I

I 20 I

In A first group (I), gag 1 comprises peptides. These peptides recognize an- I

In ten HIV-2 antibodies and are thus capable of detecting an infection with I

In HIV-2. They also recognize to some extent anti-HIV-1 antibodies. IN

Another group (II) comprises peptides which are more specifically similar to those I

In peptides located in the transmembrane portion and at the end of the I

In the outer part of the coat protein. These proteins are designated above

env1. env2 and env3. They enable a specific recognition of presence

In the journey of antibodies against HIV-2, thus allowing discrimination against I

10 limb transient or present infections due to HIV in a person, I

Specifically, a discrimination between infections that was provocative

In the course of HIV-2 and infections provoked by HIV-1. IN

In particular, the invention relates to:

I 15 I

In 1) envelope proteins and glycoproteins encoded by the env gene. and as you

1 is shown in FIG. 3, I

In the 2) protein GAG shown in FIG. 4, I

In the 3) protein POL shown in FIG. 5, I

In the 4) protein Q shown in FIG. 6, I

In the 5) protein R shown in FIG. 7, I

In the 6) protein X shown in FIG. 8, I

In the 7) protein F shown in FIG. 9, I

In the 8) protein TAT shown in FIG. 10. I

I 25 I

In the cDNA sequences encoding the above proteins are shown in FIG. IN

I 1B. The invention is in addition to the above-mentioned nucleic acid sequences en-I

In each modified nucleic acid sequence, which also encodes the proteins I

I from SIV retrovirus or a variant thereof. IN

I 30 I

I These cDNA sequences are located by the numbering shown on I

In the previously described sequences (Figure 1B), the following are:

21 DK 175879 B1 Sequence Encoding GAG Nucleotides 551-2068 "POL," 1726-4893 ...... Q, "4826-5467" X, "5298-5633 5 - ...... "R," 5637-5939 ...... F, "8569-9354 ........ TAT-1," 5788-6084 ..... ART-1, "6014-6130 .. ...... TAT-2, "8296-8391 10 - ART_2" 8294-8548 "ENV," 6090-8732

Claims (5)

1. Nucleotide sequence, characterized in that it comprises all or part of the nucleic acid sequence defined in FIG. 1B. IN 2. Nucleotide sequence, characterized in that it comprises all or part of the nucleic acid sequence shown in FIG. 1C. IN A nucleic acid sequence according to claim 1, characterized in that it comprises the nucleotide sequences: I GAG located between nucleotides 550-2068 POL 1726-4893 I 15 Q 4826-5467 I X 5298-5633 I R 5637-5939 I F 8569 - 9354 I TAT-1 5788 - 6084 I 20 ART-1 6014-6130 I TAT-2 8296 - 8391 I ART-2 8294 - 8548 I LTR 8950-9468 and I 1-316 I 25 ENV 6090 - 8732 I A recombinant nucleic acid, characterized in that it comprises all of I or a portion of a nucleotide sequence according to any one of claims I to 1-3 inserted into a nucleic acid derived from a vector. IN 30 I Recombinant nucleic acid according to claim 4, characterized in that it is labeled. IN