Nucleotide sequences derived from the genomes of the retroviruses HIV-1, HIV-2 and SIV, and their application to the amplification of pol sequences in these viral genomes and to the in vitro diagnosis of infections resulting from these viruses

IS 2 262 166 T3

DESCRIPTION

Synthesis of proteins or polypeptides encoded by an HIV-1, HIV-2 or SIV nucleotide sequence.

The present invention relates to nucleotide sequences that can be used to carry out amplification techniques for specific nucleic sequences of human immunodeficiency retroviruses of the HIV type or of the monkey immunodeficiency retrovirus of the SIV type.

The isolation and characterization of retroviruses grouped under the names HIV-1 and HIV-2 have been described in European patent applications n ° 85 / 905,513.9 (HIV-1) and n ° 87 / 400,151.4 and EP 0269520 ( HIV-2 These retroviruses have been isolated from many patients with symptoms of lymphadenopathy or acquired immunodeficiency syndrome (AIDS).

The HIV-2 type and HIV-1 type retroviruses are characterized by a tropism for human T4 lymphocytes and by a cytopathogenic effect with respect to these lymphocytes while they multiply producing, among others, generalized and persistent polyadenopathies or AIDS.

Another retrovirus, called SIV-1, this name replaces the previous name known as STLVIII, has been isolated from the rhesus macaque monkey (MD DANIEL et al. Science, 228, 1201 (1985); NL LETWIN et al. Science, 230, 71 (1985) under the name "STLV-IIImac").

Another retrovirus, named “STLV-III_AGM”(Or SIV_AGM) has been isolated from wild vervet monkeys. But contrary to what happens with the viruses present in the rhesus macaque monkey, the presence of STLV-III_AGM it does not appear to induce an AIDS-like disease in the African green monkey.

For convenience these viruses will be referred to hereinafter with the expression SIV (the expression SIV is the English abbreviation of "Simian Immunodeficiency Virus" (monkey immunodeficiency virus) eventually followed by an abbreviation that designates the species of monkey in which they are present For example "Mac" for the macaque or "AGM" for the African green monkey (short for "African Green Monkey").

A strain of retrovirus SIV-1Mac has been deposited with the CNCM on February 7, 1986 under the number I-521.

The objective of studying the HIV-1 and HIV-2 retroviruses has also led to the obtaining of complementary DNA sequences (cDNA) from the RNA of their genome. The complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-2 class (HIV-2 Rod) has been deposited with the CNCM on 2-21-1986 under number I-522, under the reference name LAV -2 Rod.

Similarly, WAIN HOBSON, SONIGO, COLE, DANOS and ALIZON in Cell (January 1985) have described the complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-1 class.

Also for convenience, viruses of the type HIV-1 and HIV-2 will sometimes hereinafter be referred to as HIV.

In vitro diagnostic procedures for infections with the type HIV-1 or HIV-2 that currently exist are based on the detection of ANTI-HIV-1 or ANTI-HIV-2 antibodies eventually present in a biological sample (biopsy ) or in a biological fluid, for example in a serum obtained, from the study patient, by putting this biological fluid in contact with extracts or antigens of HIV-1 or HIV-2, under conditions that allow the eventual production of an immunological reaction of these extracts or antigens with these antibodies.

Such diagnostic procedures have the risk of giving false negatives, particularly in the case of a recent infection of an individual with the virus of the HIV type.

Gene amplification techniques represent a considerable aid for the development of especially sensitive in vitro diagnostic procedures for viral diseases. Among these gene amplification techniques, mention may be made of the PCR (polymerase chain reaction) technique, such as that described in European patent applications No. 86 / 302,298.4 of March 27, 1986 and No. 87 / 300,203. 4 of 9-1-1987 or even the aforementioned "Q6replicase" technique described in Biotechonology, vol. 6, page 1197 (October 1988) and that which works by means of an RNA polymerase (T7RNA polymerase) described in the international patent application No. WO89 / 01050. These techniques make it possible to improve the sensitivity of detection of the nucleic acids of the viruses and require the use of specific synthesis primers.

For the search for viruses of the HIV type, the selection of primers is a problem. Indeed, due to the great variability of nucleotide sequences in the viral genome, a primer for a known sequence of a given isolate of a virus of the HIV type may fail to amplify certain viral variants of the HIV type. On the other hand, even if a primer in one conserved region of the genome of an HIV virus is chosen for another, its "good performance" is not assured and can lead to poor amplification yields.

The present invention provides precisely oligonucleotide primers that allow, among others, the ampli2

ES 2 262 166 T3 fication of all viruses of the HIV and SIV type, with yields considered as maximum in the current state of the art and, above all, avoiding the presence of numerous specific bands.

The primers used in the present invention are both specific for the viruses of the HIV-1 genome and / or for the viruses of the HIV-2 and SIV groups and are insensitive to variations in the genome of these viruses.

The present invention has as its object the use of oligonucleotide primers of approximately 15 to 30 nucleotides, usable for the genomic amplification of viruses of the HIV-1 type and / or of the HIV-2 and SIV type.

The invention applies any nucleotide sequence characterized in that its sequence:

- or it is chosen from those that are contained in one of the nucleotide sequences that comprise the gag gene of the viruses HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod and SIV Mac, and more particularly between those contained in the nucleotide sequences defined below,

- or (especially for the longer sequences) contains one of the nucleotide sequences mentioned above from HIV-1 Bru or HIV-1 Mal or HIV-1 Eli or HIV-2 Rod or SIVMac, or contains a nucleotide sequence complementary to one of these last sequences, it being understood that the possible supplementary nucleotides that "overflow" the nucleotide sequence of the genus in question, on the side of the 3 'or 5' ends, they preferably coincide with those that are located at the 5 'or 3' ends corresponding to the same sense of the complete sequence of the previously mentioned HIV-1, HIV-2 or SIV Mac viruses,

- or if this nucleotide sequence is not identical to one of the aforementioned nucleotide sequences, or is not complementary to one of these sequences, it is at least capable of hybridizing with a nucleotide sequence from the HIV-1 Bru, HIV-1 Mal virus , HIV-1 Eli and / or with a nucleotide sequence from the HIV2 Rod or SIV Mac virus mentioned above. Hybridization can be carried out at a temperature of 60 ° C ± 1 ° C, (preferably 60 ° C ± 0.5 ° C) for maximum performance.

The numbering of the nucleotides mentioned below corresponds to that used in the reference manual "Human Retrovirus and AIDS 1989" edited by the "Los Alamos National Laboratory New Mexico, USA".

The sequences of the HIV-1 Mal and HIV-1 Eli viruses have been described by MONTAIGNER, SONIGO, WAINHOBSON and ALIZON in European patent application No. EP 0 253 701.

The sequences of the invention are synthesized in a synthesizer available from Applied Biosystems (phosphoramidite method) and in any other apparatus that uses a similar procedure.

The invention relates more especially to the use of the nucleotide sequences characterized by the following nucleotide chainings (represented in the 5 '-> 3' senses; the initials "S" and "AS" indicate whether the oligonucleotide is sense or antisense, ie that is, if the oligonucleotide is oriented respectively in the 5 'or -> 3' direction or in the 3 'or -> 5' direction):

1 °) the sequences common to the genomes of the HIV-1, HIV-2 and SIV viruses (the series of figures spaced with a line indicate the position of the nucleotides on the genomes corresponding to the viruses HIV-1, Bru, HIV- 1 Mal, HIV-1 Eli, HIV-2 Rod and SIV):

• specific sequences of the gag gene of the genome of the aforementioned viruses (gene that codes for a group of specific antigens of the nucleoid of these viruses).

Certain variants can be introduced on certain positions of the nucleotide sequences mentioned below, without the hybridization properties of these nucleotide sequences with the genes of the viruses of the HIV and / or SIV type being affected. The nucleotide sequences carried by these variants are represented below the initial nucleotide sequences from which they are derived by substitution of one or more bases. The modified bases in relation to those of the initial nucleotide sequences are indicated in vertical capital letters with the positions corresponding to the bases that have been substituted in these initial sequences, while the bases of the initial sequences that have not been able to be replaced in the initial sequences. sequences carrying these variants are indicated by dots.

Primer synthesis is done using all variants simultaneously. The tests use the mixture of all variants for a given sequence.

IS 2 262 166 T3

MHyl: TGG CGC CCG AAC AGC GAC

....... T ..........

5, 636-653, 635-652, 636-653, 859-876, 834-851 MMy2 í GGC CAG GGG GAA AGA AAA A

... .C. »C ...........

.......TO. ..........

S, 854-872,864-888,848-872,1160-1184, 1124-1148 MMy3; TGC CCA TAC AAA ATG TTT TA

...... C .. TT ... ... ..

AS, 900-881,916-897,900-881,1212-1193,1176-1157

MMy4: TGC ATG GCT GCT TGA TG ..... A ..... C · .G ..

AS, 1385-1369,1419-1403,1385-1369,1703-1687,1667-1651 MMy4B: CTT TGC ATG GCT GCT TGA TG, .C ..... A ..... C. .G. .

AS, 1388-1369, 1421-1403, 1388-1369, 1706-1687,

1670-1651,

MMy4Bbis: CAT CAA GCA GCC ATG CAA AG • C. . G ... . T, G.

S, 1369-1388, 1403-1421, 1369-1388,

1687-1706, 1651-1670,

MMy2S: AGG GCT GTT GGA AAT GTG G .............. G ....

S, 2021-2039, 2055-2073, 2024-2042, 2329-2349,. 2299-2318,

MMy28bis: CCA CAT TTC CAG CAT CCC T • «♦» «» ♦ - * · - * t, · G · «» ♦

.............. C ·. · «

AS, 2039-2021, 2073-2055, 2042-2024, 2349-2329, 2318-2299

Another object of the invention is the use in the synthesis process of sequences (or primers) that have a nucleotide structure complementary to those of the primers defined below.

The invention also relates to the use in the process of synthesis of nucleotide sequences exhibiting certain mutations in relation to those defined above without modifying the hybridization properties, such as those defined above, of these sequences. The percentage of nucleotides different from those that constitute the sequences described above, without affecting the hybridization properties of the sequences of the invention, can reach up to 40%.

In general, in the case of a primer (first) in sense (S), a greater number of mutations will be tolerated at the 5 'end than at the 3' end of the primer, the 3 'end having to hybridize perfectly with a strand determined of a nucleotide sequence to allow amplification of this sequence. In the case of an antisense primer (AS), tolerance is allowed at the 3 'end.

IS 2 262 166 T3

The primers as defined above can comprise a conservation of at least 5 bases on each side, the intermediate part being the one that carries the modifications, without the previous hybridization properties being modified.

One of the characteristics of the oligonucleotide primers used in the invention is to give a clear amplification band, generally devoid of non-specific bands, using the technical indications for use described in the present invention. This fact is due to the length of the primers, which can have up to 27 bases, which increases the specificity of hybridization, as well as the drastic conditions of use that allow the elimination of parasitic associations. The specificity for each type of virus is a function, in addition to the percentage of homology with the reference matrix, of the length of the primers, which can reach up to 40 bases for an acceptable yield.

The invention also relates to the use of primers such as those described above, linked at their 5 'end to a promoter to carry out a genomic amplification procedure by means of the synthesis of multiple copies of DNA or RNA, as described in European Patent Application No. 88 / 307,102.9 of 18-1988.

The object of the invention is clearly the use of the primers described above for carrying out a procedure for gene amplification of nucleic sequences of the HIV-1 and / or HIV-2 type virus, and / or SIV. The gene amplification procedure mainly comprises the following steps:

- a step of extraction of the nucleic acid to be detected that belongs to the genome of the virus of the HIV-1, HIV2 or SIV type possibly present in the aforementioned biological sample, and if necessary, a treatment step by means of a reverse transcriptase of said acid nucleic acid, if the latter is in the form of RNA, in order to obtain a double-stranded nucleic acid (the latter stage being designated as the reverse transcription stage of the viral RNA),

- a cycle comprising the following stages:

• denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid, • hybridization of each of the nucleic acid strands, obtained during the preceding denaturation step, with at least one primer according to the invention, by contacting the aforementioned strands with at least one pair of primers according to the invention under the hybridization conditions defined below, • the formation from the primers of the complementary DNAs of the strands on which they hybridize in the presence of a polymerization agent (DNA polymerase) and of four different nucleoside triphosphate (dNTP), which leads to the formation of a greater number of double-stranded nucleic acids to be detected that in the preceding denaturation step, repeating this cycle a certain number of times to obtain said nucleic sequence to be detected, possibly present in the biological sample in a sufficient proportion to allow its detection.

The hybridization step described above is advantageously carried out at 60 ° C for 1 minute and 30 seconds in "10 X buffer", the composition of which (in final use concentration) is indicated below.

Indeed, the genomes of the HIV and SIV viruses are presented in the form of RNA or DNA depending on the location of the virus in the body.

When the virus is located inside the cells of the body, specifically inside the blood cells, its RNA is copied back into DNA by means of a reverse transcriptase. On the contrary, the genome of the HIV-type virus in extracellular medium, specifically in the blood, is in the form of RNA.

The stage of extraction of the viral DNA contained in the cells of the biological sample recommended by the inventors, different from the classical procedure of chloroform phenol, has the following stages:

• suspension of cell debris in 0.5 ml of pyrolyzed water in a Potter pacifier.

• “round trip” disruption of cells, • addition of Triton X100 for 1 final concentration of 0.1%, • heat denaturation for 15 to 25 minutes at 100 ° C, • short centrifugation to remove cellular debris,

ES 2 262 166 T3 • DNA precipitation overnight at -20 ° C adding 2.5 volumes of absolute ethanol and 10% of the final volume of 3 molar sodium acetate. The DNA is then recovered, then resuspended in water and after having been washed twice with ethanol at 70 ° C together the DNA and RNA, which allows the detection of the genomic message of the viruses of the HIV and SIV types. using the procedure called "PCR direct DNA" or by the so-called "PCR-RNA".

The viral RNA extraction step is generally carried out according to the classical procedure known to those skilled in the art.

After the extraction of the RNA, it is necessary to carry out an additional step of transformation of the single-stranded RNA into double-stranded DNA, while the in vitro diagnosis of the invention is carried out from biological samples containing the viruses of the HIV-type. 1 and / or HIV-2 and / or SIV, whose genomes are in RNA form.

This transformation of RNA into DNA is carried out by treating the RNA obtained after extraction of the biological sample, especially the serum, in an appropriate medium by means of a reverse transcriptase.

The reverse transcription stage of the viral RNA is carried out as follows:

-10 pg of the extracted RNA resuspended in water is placed in the presence of a pair of primers with a concentration of 40 pM each, in a final volume of 40 µl.

The whole is denatured at 100 ° C for 10 minutes, then immersed in ice water,

- Add 10 μl of the following mixture: 5 μl of the buffer "10 X buffer" described below + 1 unit of reverse transcriptase (from AMV (Avian Myeloblastosis Virus) or from MuMLV (Moloney Leukemia Virus)) + 1 unit of Taq polymerase +1 µl of mixture of the 4 dNTPs, at a rate of 25 mM each + water qs 10 µl. The final volume is 50 pl.

This reaction takes place in two stages:

- a) first step: manufacture of the cDNA by the action of reverse transcriptase at 42 ° C for 13 minutes,

- b) second step: classical gene amplification: heat at 95 ° C for 3 minutes to destroy the reverse transcriptase and allow the dehybridization / hybridization step, then the cycle described above for gene amplification begins.

The denaturation step is carried out in the presence of a primer (s) (or primers) of the invention. Indeed, as specified above, one of the characteristics of the oligonucleotides (or primers) of the invention is to give a clear amplification band, generally devoid of specific bands, being used under the following conditions:

hybridization: the primers (1 µl of a 40 pmolar solution) of each primer) are placed in the presence of the template DNA (100 to 300 ng) for the first denaturation-annealing step; It is heated for 10 minutes at 100 ° C, then the tubes containing this DNA-template mixture and the primers are immersed in the ice water in order to increase the reassociation rate of the template / primer DNA. The primers should be used at a final concentration in the next amplification step of 0.8 pm each.

- amplification: the 4 dNTPs are added to the above medium, each of them at 0.5 pmolar in final solution (50 µl) and one unit of Taq-polymerase for a reaction medium of 50 µl; This step is carried out in an amplification buffer of the present invention generally called "10 X buffer", whose composition (when diluted to 1/10) is the following: Tris-HCl, pH 8.9: 50 mM; (NH₄)₂SW₄: 15 mM; MgCl₂: 5 mM; ^ - mercapto-ethanol: 10 mM, gelatin: 0.25 mg / ml. 5 µl of this buffer and water are added qs 50 µl to the previous medium.

The amplification cycles are carried out as follows: 30 to 40 cycles composed of:

94 ° C for 10 seconds (denaturation),

60 ° C for 1 minute 30 (hybridization),

78 ° C for 1 minute 30 (elongation).

This is followed by a single cycle at 78 ° C for 15 minutes.

The precision of the temperatures indicated at approximately ± 0.3 ° C, as well as their stability during the different cycles, represent essential conditions for obtaining maximum yields, as well as the absence of specific bands.

IS 2 262 166 T3

The optimal DNA concentration is 100 to 300 ng for genomic DNA extracted from cells (from patients or in culture, from mammals or others).

It is understood that the above conditions represent optimal conditions for a final reaction medium of 50 joule and that these conditions can be modified as a function of the final volume of the reaction medium.

The polymerization agent used in the cycle elongation step is a thermostable DNA polymerase, specifically Taq polymerase, the amplificase from Appligéne company as commercially available thermostable DNA polymerase.

In general, the cycle of the gene amplification procedure of the invention is repeated between 30 and 40 times.

Exemplary primer pairs for the gene amplification procedure are as follows:

- MMy1-MMy4, MMy2-MMy4, MMy1-MMy3, MMy4bis-MMy28bis for the gag gene.

In any case, the combinations between primers "S" and "AS" described above do not represent limitations and can be varied according to the desire of the user.

In the following table, by way of example, the dimensions of the nucleotide fragments synthesized with the aid of the aforementioned primer pairs are indicated.

(The figures indicated in the following tables represent the number of nucleotides of the synthesized fragments and "the lines" indicate that the pairs of primers tested do not allow the characterization of the corresponding viral strains.

TABLE I

<td colspan="3">gag</td><td colspan="2">gag</td>

<td colspan="5">: MMy1-MMy3: MMy1-MMy4: MMy4bis-MMy28bis:</td>

<td>HIV1-BRU:</td><td> 265</td><td> 750</td><td> 532</td><td> 671</td>

<td>HIV1-MAL:</td><td> 282</td><td> 785</td><td> 556</td><td> 671</td>

<td>HIV1-ELI:</td><td> 265</td><td> 750</td><td> 538</td><td> 674</td>

<td>HIV2-ROD:</td><td> 354</td><td> 845</td><td> 544</td><td> 663</td>

<td>SIV:</td><td> 343</td><td> 844</td><td> 544</td><td> 668</td>

Another object of the invention is the application of oligonucleotides such as those described above and which include sugars in the α-conformation. Said oligonucleotides have the characteristic of reversing the sense of the double helix formed with the template (virus genome strand), thus passing this double helix from the "S" state to the "AS" state.

The invention also relates to the application of the oligonucleotides described above, among which some nucleotides are methylated and / or have one or more sulfur atoms, specifically on adenines. Such oligonucleotides have the characteristic of increasing the stability of the double helix and, consequently, they hybridize better with the DNA strand to be amplified.

The invention also refers to the application of oligonucleotides such as those described above and which are in the form known as "modified bases", which have nucleotides on which chromophore agents are covalently bound (flat aromatic molecules, such as orange acridine), specifically according to the procedure described in the article by C. Hélene published in "La vie des Sciences", general series, volume 4, n ° 1, page 17-37. Said oligonucleotides have the characteristic of being easily detectable, specifically by fluorescence.

The invention also relates to the use of the primers of the invention indicated above for carrying out a process for the synthesis of proteins encoded by the nucleotide sequences amplified by means of these primers.

IS 2 262 166 T3

The process for synthesizing a protein or a polypeptide encoded by a nucleotide sequence of the HIV-1, HIV-2 or SIV virus according to the invention is characterized in that it comprises the following steps:

to. synthesis of the nucleotide sequence by a procedure of gene amplification of nucleotide sequences of viruses of the type HIV-1, HIV-2 or SIV, with the help of at least two ligonucleotide primers whose sequences each consist of

i. a specific sequence of the gag gene chosen from a conserved region between the genomes of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod and SIV Mac viruses capable of hybridizing at a temperature of 60 ° C ± 1 ° C with the genomes of the viruses HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod and SIV Mac or ii. A sequence complementary to a sequence such as that defined in i .; Y

b. Recovery of the nucleotide sequence as well as amplified and translation into protein.

A particular synthesis procedure is one in which the sequence of the primers has at least 60% identity with the sequence of the gag gene of an HIV-1 Bru, or HIV-1 Mal, or HIV-1 Eli virus. or HIV-2 Rod or SIV Mac.

Another particular synthesis method according to the invention is characterized in that the oligonucleotide primers comprise a conservation of at least 5 bases on each side of the primer with respect to the sequence of the gag gene of an HIV-1 Bru, HIV-1 Mal, HIV-1 virus Eli, or HIV-2 Rod or SIV Mac, and retain the properties of hybridization with the genomes of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod and SIV Mac viruses.

Another method according to the invention is characterized in that the gene amplification process comprises the following steps:

to. a step of extraction of the nucleic acid that belongs to the genome of the virus of the type HIV-1, HIV-2 or SIV, and optionally a step of treatment by means of a reverse transcriptase of said nucleic acid, if the latter is in the form of RNA.

b. a cycle comprising the following stages:

i. - denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid, ii. - hybridization of each of the nucleic acid strands, obtained during the preceding denaturation step, with at least one nucleotide primer by contacting the aforementioned strands with at least one pair of primers according to one of claims 1 or 2, iii. - formation, from the primers, of the complementary DNAs of the strands on which said primers hybridize in the presence of a DNA polymerase and four different nucleoside triphosphate (dNTP), which leads to the formation of a greater number of nucleic acids double-stranded than in the preceding denaturation step, this cycle being repeated a number of times determined to obtain said nucleic sequence in a sufficient proportion to allow its detection.

Another method according to the invention is characterized in that the at least two oligonucleotide primers have sequences each consisting of:

IS 2 262 166 T3

i.- at least one sequence chosen from the following group of sense sequences:

S'-TGG CGC CCG AAC AGG GAC-3 '

5'-TGG CGC CTG AAC AGG GAC-3 'δ'-GGC CAG GGG GAA AGA AAA A-3'

<td>-GGC</td><td>CCG</td><td>GCG</td><td>GAA</td><td>AGA</td><td>AAA</td><td>A-3 '</td>

<td>-GGC</td><td>CAG</td><td>GAG</td><td>GAA</td><td>AGA</td><td>AAA</td><td>A-3 '</td>

<td>-CAT</td><td>CAA</td><td>GCA</td><td>GCC</td><td>ATG</td><td>CAA</td><td>AG-3</td>

<td>-CAC</td><td>CAG</td><td>GCA</td><td>GCT</td><td>ATG</td><td>CAG</td><td>AG-3</td>

<td>-AGG</td><td>GCT</td><td>GTT</td><td>GGA</td><td>AAT</td><td>GTG</td><td>G-3 '</td>

<td>-AGG</td><td>GCT</td><td>GTT</td><td>GGA</td><td>AGT</td><td>GTG</td><td>G-3 '</td>

and at least one sequence chosen from the following group of antisense sequences:

3'-TGC CCA TAC AAA ATG TTT TA-5 '' 3 '-TGC CCA CAC TAT ATG TTT TA-5' 3'-TGC ATG GCT GCT TGA TG-5 '

<td>-TGC</td><td>ATA</td><td>GCT</td><td>GCC</td><td>TGG</td><td>TG-5</td><td>i '</td>

<td>-CTT</td><td>TGC</td><td>ATG</td><td>GCT</td><td>GCT</td><td>TGA</td><td>TG-5</td>

<td>-CTC</td><td>TGC</td><td>ATA</td><td>GCT</td><td>GCC</td><td>TGA</td><td>TG-5</td>

<td>-CCA</td><td>CAT</td><td>TTC</td><td>CAG</td><td>CAT</td><td>CCC</td><td>T-5 '</td>

<td>-CCA</td><td>CAT</td><td>TTC</td><td>CAG</td><td>CAG</td><td>CCC</td><td>T-5 '</td>

<td>-CCA</td><td>CAT</td><td>TTC</td><td>CAG</td><td>CAC</td><td>CCC</td><td>T-5 '</td>

ii. - a sequence complementary to a sequence such as that defined in i .; or iii. - a sequence that has at least 60% identity with one of the sequences defined in io ii and capable of hybridizing at a temperature of 60 ° C ± 1 ° C with the genomes of the viruses HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod, and SIV Mac.

Another method according to the invention is characterized in that at least two of the following primer mixes are used for amplification:

MMy1: mixture consisting of primers

<img file="ES2262166T3_D0001.tif" />

IS 2 262 166 T3

MMy2: Mixture consisting of primers δ'-GGC CAG GGG GAA AGA AAA A-3 '5' -GGC CCG GCG GAA AGA AAA A-3 '5' -GGC CAG GAG GAA AGA AAA A-3 '

MMy3: mixture consisting of primers

3'-TGC CCA TAC AAA ATG TTT TA- 5 '' 3'-TGC CCA CAC TAT ATG TTT TA-5 '

MMy4: mixture consisting of primers

3'-TGC ATG GCT GCT TGA TG-5 '3'-TGC ATA GCT GCC TGG TG-5¹

MMy4B: Primer mixture

3'__{CTT TGC ATQ GCT QCT TGA tg}__5j3'-CTC TGC ATA GCT GCC TGA TG-5 '

MMy4Bbis: Primer mixture

5'-CAT CAA GCA GCC ATG CAA AG-3¹5'-CAC CAG GCA GCT ATG CAG AG-3 '

MMy28: mixture consisting of primer

5'-AGG GCT GTT GGA AAT GTG G-3 '5'-AGG GCT GTT GGA AGT GTG G-3'

MMy28bis: Primer mixture

3'-CCA CAT TTC CAG CAT CCC T-5 '3'-CCA CAT TTC CAG CAG CCC T-5' 3'-CCA CAT TTC CAG CAC CCC T-5 '

IS 2 262 166 T3

Another method according to the invention 12 is characterized in that it is carried out under the following conditions:

- for the hybridization step: 1 µl of a 40 // molar solution of each primer is placed in the presence of 100 to 300 ng of template DNA for the first denaturation-reassociation step; It is heated for 10 minutes at 100 ° C, and then the tubes containing this DNA-template mixture and the primers are immersed in the ice water, the primers being used in a final concentration in the next amplification step of 0.8 pm each.

- for the amplification step: the 4 dNTPs are added to the previous medium, each of them used at 0.5 // molar in 50 // l of final solution and one unit of Taq-polymerase for a reaction medium of 50 //1; This step is carried out in the amplification buffer called "10 X buffer", which comprises when diluted 1/10 in the final solution: Tris-HCl, pH = 8.9: 50 mM; (NH₄)₂SW₄; 15 mM; MgCl₂; 5 mM; jd-mercaptoethanol; 10 mM; gelatin: 0.25 mg / ml.

Another method according to the invention is characterized in that the translation step is carried out by transforming appropriate host cells using vectors containing said amplified sequences and recovering the proteins produced in these host cells.

Another method according to the invention is characterized in that the oligonucleotide primers are chosen from the following pairs of primer mixtures:

to. MMy1-MMy4

MMy1: mixture consisting of primers

5'-TGG CGC CCG AAC AGG GAC-3 '5' -TGG CGC CTG AAC AGG GAC-3 '

MMy4: mixture consisting of primers

3'-TGC ATG GCT GCT TGA TG-5 '3'-TGC ATA GCT GCC TGG TG-5'

b. MMy2-Mmy4

MMy2: mixture consisting of primers

5'-GGC CAG GGG GAA AGA AAA A-3 '

5 '-GGC CCG GCG GAA AGA AAA A-3'

5 '-GGC CAG GAG GAA AGA AAA A-3'

MMy4: mixture consisting of primers

3'-TGC ATG GCT GCT TGA TG-5 '3' -TGC ATA GCT GCC TGG TG-5 '

IS 2 262 166 T3

c. MMy1-MMy3

MMy1: mixture consisting of primers

5 '^ TGG CGC CCG AAC AGG GAC-3' 5'-TGG CGC CTG AAC AGG GAC-3 '

MMy3: mixture consisting of primers

3 * -TGC CCA TAC AAA ATG TTT TA-5 3'-TGC CCA CAC TAT ATG TTT TA-5 '

d. MMy4Bbis-MMy28bis

MMy4B: Primer mixture

3'-CTT TGC ATG GCT GCT TGA TG-5 '3' -CTC TGC ATA GCT GCC TGA TG-5 '

MMy28bis: Primer mixture

3'-CCA CAT TTC 3 '-CCA CAT TTC 3'-CCA CAT TTC

AGC CAT CCC T-5 'AGC AGC CCC T-5' AGC CAC CCC T-5 '

The last stage of translation is carried out especially by transforming appropriate host cells with the aid of vectors containing said amplified sequences, and recovering the proteins produced in these host cells.

The invention also concerns the polypeptides obtained from the translation of the nucleotide sequences (or primers) of the invention.

The present application also describes immunogenic compositions containing one or more translation products of the nucleotide sequences amplified according to the procedures described above from the primers defined according to the invention, translation products associated with a pharmaceutically acceptable vehicle.

The present application describes antibodies directed against one or more of the translation products described above (or in other words, capable of forming an immunological reaction with one or more translation products of the nucleotide sequences according to the invention, or even one or more translation products of the nucleotide sequences amplified from the primers defined according to the invention).

A process for preparing the nucleotide sequences or (primers) described above comprises the following steps:

- incubation of genomic DNA, isolated from one of the previously mentioned HIV or SIV type viruses, with DNase I, followed by addition of EDTA and purification by extraction in the phenol / chloroform / isoamyl alcohol mixture (25/24 1 ) and then in ether,

IS 2 262 166 T3

- treatment of the DNA thus extracted with Eco R1 methylase in the presence of DTT and purification by extraction as described above,

- incubation of the DNA thus purified with the 4 deoxynucleotides triphosphate dATP, dCTP, dGTP and dTTP in the presence of T4 DNA polymerase and DNA ligase from E. coli, followed by purification according to the procedure described above,

- the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid investigated by means of an appropriate probe.

An especially advantageous process for the preparation of the nucleotide sequences of the invention comprises the following steps:

- DNA synthesis using the automated procedure of ^ -cyanthyl phosphoramidite described in Bioorganic Chemistry 4; 274-325 (1986),

- the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid by hybridization with an appropriate probe.

Another process for preparing the nucleotide sequences of the invention comprises the following steps:

- the assembly of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences being compatible with the amino acid chain of the natural polypeptide according to the principle described in Proc. Natl. Acad. Sci. USA, 80; 7461-7465 (1983),

- the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid investigated by hybridization with an appropriate probe.