FR2647809A1 - OLIGONUCLEOTIDE PRIMERS FOR THE AMPLIFICATION OF THE GENOME OF HIV-1, HIV-2 AND SIV RETROVIRUSES AND THEIR APPLICATIONS TO IN VITRO DIAGNOSIS OF INFECTIONS DUE TO THESE VIRUSES - Google Patents

Abstract

The present invention relates to nucleotide sequences derived from the genomes of viruses of the HIV-1 type, or from the genomes of viruses of the HIV-2 type, or of viruses of the SIV type, and their applications, in particular as oligonucleotide primers for the preparation. implementation of a method for the in vitro diagnosis of the infection of an individual by a virus of the HIV-1 and / or HIV-2 type.

Description

OLIGONUCLEOTIDE PRIMERS FOR THE AMPLIFICATION OF THE GENONE OF HIV-1, HIV-2 AND SIV RETROVIRUSES,
AND THEIR APPLICATIONS TO IN-VITRO DIAGNOSIS OF
INFECTIONS DUE TO THESE VIRUSES.

 The present invention relates to oligonucleotide sequences which can be used for the implementation of techniques for amplification of specific nucleic sequences of human immunodeficiency retrovirus of the HIV type or of monkey immunodeficiency retrovirus of the SIV type.

The invention relates in particular to the application of these primers to methods of in vitro diagnosis in humans of the infection of an individual by a retrovirus of the HIV type (currently HIV-1 and / or
HIV-2).

The isolation and characterization of retroviruses grouped under the designations HIV-1 and HIV-2 have been described in European patent applications No. 85 / 905.513.9 and No. 87 / 400.151.4 respectively. These retroviruses have been isolated from several patients with symptoms of lymphadenopathy or
Acquired Immunodeficience Syndrome (AIDS).

HIV-2 type retroviruses, like HIV-1 type retroviruses, are characterized by a tropism for human T4 lymphocytes and by a cytopathogenic effect with respect to these lymphocytes when they multiply therein, thereby causing either generalized and persistent polyadenopathies, i.e.
AIDS.

Another retrovirus, called SIV-1, this name replacing the previously known name STLV-III, was isolated in 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, designated "STLV-IIIA>", (or SIVA>) was isolated from wild green monkeys. But unlike the viruses present in the rhesus macaque monkey, the presence of STLV-IIIAz does not seem to induce an AIDS-type disease in the green African monkey.

For the convenience of language, these viruses will no longer be designated in what follows only by the expression SIV (the expression SIV is the English abbreviation for "Simian
Immunodeficency Virus "(monkey immunodeficiency virus) possibly followed by an abbreviation designating the species of monkey from which they come, for example" MAC "for the macaque" or "AGM" for the African green monkey (abbreviation of "African Green Mon) cey").

 A strain of the SIV-1Mac retrovirus was deposited at the C.N.C.M on February 7, 1986 under the number I-521.

Continuation of the study of HIV-1 retroviruses and
HIV-2 has also led to the production of complementary DNA sequences (cDNAs) of the RNAs of their genome. The complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-2 class (HIV-2 ROD) was deposited on 02/21/1986 at the CNCM under the number I-522, under the reference name LAV-2 ROD.

Likewise, the complete nucleotide sequence of a
CDNA of a representative retrovirus of the HIV-1 class is described by WAIN -HOBSON, SONIGO, COLE, DANOS and
ALIZON in CEll (January 1985).

 Also for the convenience of language, viruses of the HIV-1 and HIV-2 type will sometimes be designated in the following by the expression HIV.

The methods of in vitro diagnosis of infections with viruses of the HIV-1 or HIV-2 type currently existing, call for the detection of antibodies.
ANTI-HIV-1 or anti-HIV-2 possibly present in a biological sample (biopsy) or in a biological fluid, in particular in a serum obtained, from the patient under study, by bringing this biological fluid into contact with extracts or antigens of HIV-1 or HIV-2, under conditions allowing the production of a possible immunological reaction of these extracts or antigens with these antibodies.

Such diagnostic methods risk being falsely negative, especially in the case of a recent infection of an individual with viruses of the type
HIV.

Gene amplification techniques are a considerable supplement to the development of particularly sensitive in vitro diagnostic methods for viral diseases. Among these gene amplification techniques, one can cite the PCR technique (Polymerase
Chain Reaction) as described in the European patent applications n 86 / 302.298.4 of 03/27/1986 and n 87 / 300.203.4 of 09/01/1987, or also the technique called "Qssreplicase" described in Biotechnology, vol. 6, page 1197 (October 1988) and that using a
RNA polymerase (T7RNA polymerase) described in international patent application no W089 / 01050. These techniques make it possible to improve the detection sensitivity of nucleic acids of viruses, and require the use of specific synthetic primers.

For the detection of HIV type viruses, the choice of primers is problematic. In fact, due to the great variability of the nucleotide sequences of the viral genome, a primer conforming to the known sequence of a given isolate of an HIV type virus may fail to amplify certain viral variants of the type
HIV. On the other hand, even if a primer is chosen in a conserved region of the genome of an HIV virus to another, its "good functioning" is not guaranteed and can give rise to poor amplification yields.

 The present invention precisely provides oligonucleotide primers allowing the amplification, in particular for diagnostic purposes, of the genome of all viruses of the HIV and SIV type, with good yields.

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

 The present invention relates to oligonucleotide primers, of approximately 15 to 30 nucleotides, which can be used for genomic amplification of viruses of the HIV-1 type and / or of the HIV-2 and SIV type.

 The invention relates to any nucleotide sequence characterized in that its sequence - either is chosen from those which are contained in one of the nucleic sequences included in the gag, env, nefi, nef2, vifl, vpr, vif2, vpx genomes genes HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod, or SIV-MAC viruses.

- Either (in particular for the longest primers) contains one of the abovementioned nucleic sequences derived from HIV-1 Bru, or HIV-1 Mal, or HIV-1 Eli or HIV-2 ROD or SIVMac, or contains a nucleic sequence complementary to one of these latter sequences, it being understood that the optional additional nucleotides which "extend" over the nucleic sequence of the genus in question, on the 3 ′ or 5 ′ end side, preferably coincide with those which are placed below 5 'or 3' ends corresponding within the complete sequence of viruses of the type
HIV-1, HIV-2 or SIV MAC, mentioned above, - either, if the sequence of this primer is not identical to one of the above nucleic sequences, or is not complementary to one of these sequences, is nevertheless capable of hybridizing with a nucleic sequence derived from the HIV-1 Bru, HIV-1 Mal viruses,
HIV-1 Eli, and / or with a nucleic sequence derived from the HIV-2 ROD or SIV MAC virus mentioned above.

Hybridization can be carried out at temperatures of 60'-55'C, the temperature recommended for optimum performance.

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

(The sequences of the HIV-1 Mal, HIV-1 Eli viruses have been described by MONTAGNIER, SONIGO, WAIN-HOBSON and
ALIZON).

 The primers are synthesized on a synthesizer marketed by Applied Biosystems (phosphoro-amidites method).

The invention relates more particularly to the oligonucleotide sequences characterized by the following nucleotide sequences (represented in the 5 ′ - 3 ′ direction; the initials “S” and “AS” indicate whether the oligonucleotide is sense or antisense, ie say whether the oligonucleotide is oriented respectively in the 5 '- 3' direction or in the 3 '- 5' direction) 1) sequences common to the genomes of the HIV-1 viruses,
HIV-2 and SIV (the series of figures spaced by a line indicate the position of the nucleotides on the genomes, corresponding respectively to the HIV-1 viruses
Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV)
specific sequences of the gag gene of the genome of the above-mentioned viruses (gene coding for a group of antigens specific to the nucleoid of these viruses).

MMyl: TGG CGC CCG AAC AGG GAC
or T
: S, 636-653, 635-652, 636, 653, 859-876,
834-851
MMy2: GGC CAG GGG GAA AGA AAA A: S, 854-872,864-888,848-872,1160-1184,
1124-1148
MMy3: TGC CCA TAC AAA ATG TTT TA
or CTT AS, 900-881,916-897,900-881,1212-1193,1176-1157
MMy4: TGC ATG GCT GCT TGA TG AS, 1385-1369, 1419-1403,1385-1369, 1703-1687,1667-1651
specific sequences of the vpr gene
MMyl8: GAT AGA TGG AAC AAG CCC CAG
: S,
MMyl9: TCC ATT TCT TGC TCT CCT CTG T
: AS, 2M) sequences common to the genomes of the HIV-2 viruses and
SIV (the series of figures spaced by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-2 ROD virus, and 51V-MAC).

specific sequences of the nef2 gene (coding for a negative factor of 27 kD)
MMyl2: AGA GAC TCT TGC GGG CGC GTG
: S, 9165-9185,
MMyl3: ATA TAC TTA GAA AAG GAA GAA GG
: S, 9542-9564,
MMyl3bis: CCT TCT TCC TTT TCT AAG TAT
AS, 9564-9542,
MMyl4: AGC TGA GAC AGC AGG GAC TTT CCA
AS, 9956-9933,
specific sequences of the vif2 gene (coding for an infectivity factor of 23 kD)
MMy20: TAT GGA GGA GGA AAA GAG ATG GAT AGT
: S, MMy21: TAG GAC TTA TTT CCC TTG CTT T
: S,
MMy2lbis: AAA GCA AGG GAA ATA AGT GCT A
: AS,
MMy22: CCC TTG TTC ATC ATG CCA GTA T
: AS,
specific sequences of the vpx gene (coding for a 12 kD protein)
MMy23: ATG TCA GAT CCC AGG GAG AS,
MMy24: CCT GGA GGG GGA GGA GGA GGA
: AS, 3p) Sequences common to the genomes of HIV-1 Bru viruses,
HIV-1 Mal, and HIV-1 Eli (the series of figures spaced by a line indicate the position of the nucleotides on the genomes corresponding respectively to the viruses HIV-1- Bru, HIV-1 MA1 and HIV-1 Eli).

specific sequences of the env gene (encoding envelope proteins)
MMy5: CCA ATT CCC ATA CAT TAT TGT GCC CC
: S, 6905-6930,6903-6928,6860-6885
MMy6: AAT GGC AGT CTA GCA GAA GAA GA: S, 7055-7077,7053-7075,7010-7032
MMy7: ATC CTC AGG AGG GGA CCC AGA AAT T
: s, 7360-7384, 7349-7373,7306-7330
MMy7bis: AAT TTC TGG GTC CCC TCC TGA GGA T
: AS, 7384-7360,7373-7349,7330-7306
MMy8: GTG CTT CCT GCT GCT CCC AAG AAC CC
: AS, 7857-7832,7846-7821,7800-7775
specific sequences of the nefi gene
MMy9: ATG GGT GGC AAG TGG TCA AAA AGT AG
or A
: S, 8844-8869
MMyl0: AAA AGA AAA GGG GGG ACT GGA
: S, 9116-9136,
MMylObis: TCC AGT CCC CCC TTT TCT TTT
: S, 9136-9116
MMyll: AAA GTC CCC AGC GGA AAG TCC C AS, 9503-9483,
specific sequences of the vifl gene
MMyl5: GAT TAT GGA AAA CAG ATG GCA GGT GAT
: S,
MMyl6: GCA GAC CAA CTA ATT CAT CTG TA S,
MMyl6bis: TAC AGA TGA ATT AGT TGG TCT GC
: AS,
MMyl7: CTT AAG CTC CTC TAA AAG CTC TA
: AS,
A subject of the invention is also the primers having a nucleotide structure complementary to those of the primers defined above.

 It also relates to primers exhibiting certain mutations with respect to those defined above without the hybridization properties, as defined above, of these primers being modified. The percentage of nucleotides different from those constituting the primers described above, without however affecting the hybridization properties of the primers of the invention, can reach 40%.

 Generally, in the case of an S primer, a greater number of mutations will be tolerated on the 5 'side than on the 3' side of the primer, the 3 'side having to hybridize perfectly with a determined strand of a nucleic sequence to allow the amplification of this sequence. In the case of an anti-sense primer, tolerance is allowed on the 3 'side.

 A subject of the invention is also the primers as defined above and comprising a preservation of at least 5 bases on each side, the middle part comprising modifications, without the above hybridization properties being modified.

 One of the characteristics of the oligonucleotide primers of the invention is to give a clear amplification band, generally devoid of non-specific bands. This fact is due to the length of the primers which can reach 27 bases which increases the specificity of hybridization, as well as to the drastic conditions of use which make it possible to eliminate the 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, for an acceptable yield, up to 40 bases.

 The invention also extends to primers as described above linked at the level of their 5 ′ end to a promoter for the implementation of a method of genomic amplification by synthesis of multiple copies of DNA or of RNA as described in European patent application n 88 / 307.102.9 dated 08/01/1988.

 The invention particularly relates to the use of the primers described above for the implementation of a method of in vitro diagnosis of the potential infection of an individual by a virus of the HIV-1 and / or HIV type -2.

This in vitro diagnostic method of the invention is carried out using a biological sample (in particular a biological fluid such as serum) obtained from a patient under study, and mainly comprises the following steps - a step of extraction of the nucleic acid to be detected belonging to the genome of the virus of the HIV-1 and / or HIV-2 type possibly present in the abovementioned biological sample, and, where appropriate, a step of treatment with using a reverse transcriptase of said nucleic acid if the latter is in the form of RNA in order to obtain a double-stranded nucleic acid, - a cycle comprising the following steps
denaturation of the double-stranded nucleic acid & detect, 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 bringing the above-mentioned strands into contact with at least one pair of primers according to invention under the hybridization conditions defined above,
elongation of the primers along the strands on which they are hybridized in the presence of a polymerization agent and four different nucleoside triphosphate (dNTP), which leads to the formation of a greater number of double-stranded nucleic acid to be detected than in the previous denaturation step, this cycle being repeated a number of times determined to obtain said nucleic sequence to be detected, possibly present in the biological sample in a sufficient proportion to allow its detection, - a step of detecting the presence optional nucleic acid belonging to the genome of the virus of the HIV-1 and / or HIV-2 type in the biological sample.

 The in vitro diagnostic method of the invention can be carried out either from viral RNA or from complementary, episomal or integrated DNA.

 Indeed, the genomes of the HIV and SIV viruses are 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, especially inside the blood cells, its RNA is copied into DNA by reverse transcriptase. On the other hand, the genome of HIV type viruses in an extracellular medium, especially in the blood, remains in the form of RNA.

The step of extracting the viral DNA contained in the cells of the biological sample recommended by the inventors - in addition to the conventional phenol chloroform method - presents the following steps: suspension of the cell pellet in 0.5 ml of pyrolized water in a large piston Potter, grinding of the cells known as "round trip", addition of Triton X100 for 1 final concentration of 0.1%, heat denaturation for 15 minutes at 100 ° C, centrifugation to remove cellular debris,
DNA precipitation overnight at -20 ° C by adding 2.5 volumes of absolute ethanol and 10% of the final volume of 1 Molar sodium acetate. The DNA is then recovered and then resuspended in water after having been washed with 709 ethanol.

 The viral RNA extraction step is generally carried out in a conventional manner known to those skilled in the art.

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

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

 The invention more particularly relates to an in vitro diagnostic method as defined above, in which the step of reverse transcription of the viral RNA is carried out as follows - 10 Mg of extracted RNA resuspended in l water is placed in the presence of the pair of primers at a concentration of 0.8 pM each, in a final volume of 40 l.

The whole is denatured at 100-C for 10 minutes then immersed in ice water, - 10 p1 of the following mixture are added: Ctl of the buffer "10 X buffer" described below + 1 unit of reverse- transcriptase + 1 unit of Taq-polymerase + 1 Cil of the mixture of 4 dNTPs at 25 mM each + water
QSP 10 p1. The final volume is therefore 50 p1.

This reaction takes place in two stages
a) first step: manufacture of the cDNA by the action of reverse transcriptase at 42iC for 13 minutes,
- b) 2nd step: classic gene amplification: heating to 95 ° C. for 3 minutes to destroy the reverse transcriptase and allow the dehybridization / hybridization step, then the cycle described above for gene amplification is started.

 These - original conditions are part of the invention.

 One of the characteristics of the oligonucleotides presented is to give a clear amplification band, generally devoid of non-specific bands.

This fact is due to the length of primers which can reach 27 bases as far as we are concerned - which increases the specificity of hybridization, as well as the drastic conditions of use which make it possible to eliminate 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, for an acceptable yield, up to 40 bases. This therefore makes it possible to cover a certain number of variants.

 These particular conditions form an integral part of the applications that we recommend, and, consequently, of our deposit - hybridization: the primers must be placed in the presence of DNA-matrix for the first stage of denaturation-reassociation; heating, for 10 minutes at 100-C then immerse the tubes in water containing ice. The oligonucleotides should be used at a final concentration of 0.8 M each.

- amplification: the amplification buffer, generally designated under the name of "10 X buffer", which we recommend is a buffer of our manufacture which is part of this present deposit. Its composition is as follows: Tris-HCl, pH 8.9: 50 mM: (NH4) 2 SO4: 15 mM 1 MgCl2: 5 mM ss-mercapto-ethanol: 10 mM gelatin: 0.25 mg / ml.

The nature of the amplification cycles is part of the conditions optimized for a maximum yield of the oligonucleotides and are part of this deposit: 30 & 40 cycles composed of
94C for 10 seconds (denaturation),
. 60 C for 1 minute 30 (hybridization),
. 78 C for 1 minute 30 (elongation).

 Everything will be followed by a single cycle at 78 ° C for 15 minutes.

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

 The optimal hybridization buffer (designated "10 x buffer") used in the cycle hybridization step comprises: Tris-HCl, pH = 8.9: 50 mM; (NH4) 2 SO4; 15 mM; MgCl2; 5 mM; ss-mercapto-ethanol: 10 mM gelatin: 0.25 mg / ml.

The use of several different pairs of primers (or couple cocktails) of the invention allows either the cross-detection of several types of viruses of the type
HIV and / or SIV, ie the simultaneous detection of several genes of the same virus of the HIV and / or SIV type.

By way of example of preferred pairs of primers which can be used in the context of the present invention, mention may be made of the following pairs of primers -MMyl-MMy4 (650 bases), MMy2-MMy4 (538 bases), MMyl
MMy3 (265 bases), MMyl8-MMyl9 (275 bases), for the in vitro diagnosis of HIV-1 infection and / or
HIV-2 - MMy5-MMy8 (953 bases), MMy6-MMy8 (803 bases), MMy7
MMy8 (498 bases), MMyS-MMy7bis (480 bases), MMy6
MMy7bis (330 bases), MMy9-MMyll (660 bases), MMy9
MMylObis (292 bases), MMyl5-MMyl7 (600 bases), MMyl5
MMyl6bis (335 bases), MMyl6-MMyl7 (290 bases), for the in vitro diagnosis of an individual's infection by
HIV-1, - MMy20-MMy22 (655 bases), MMy20-MMy2lbis (350 bases), MMy21-MMy22 (325 bases), MMy23-MMy24 (230 bases),
MMyl2-MMyl4 (791 bases), MMyl2-MMyl3bis (600 bases), for the in vitro diagnosis of an individual's infection with HIV-2,
The polymerization agent used in the elongation stage of the cycle is a DNA polymerase, in particular Taq polymerase.

 Generally, the cycle of the in vitro diagnostic method of the invention is repeated between 30 and 40 times.

For the gene by gene diagnostic method of the invention, the pairs MMyl-MMy4, MMy2 are used.
MMy4, MMyl-MMy3 for the gag gene, MMyl8-MMyl9 for the vpr gene, MMys-MMy8, MMy6-MMy8, MMy7-MMy8, MMy5
MMy7bis, MMy6-MMy7bis, for the env gene, MMy9-MMyll,
MMy9-MMylObis for the nefl gene, MMylS-MMyl7, MMyl5
MMyl6bis, MMyl6-MMyl7, for the vifl gene, MMy20-MMy22, MMy20-MMy21bis, MMy21-MMy22 for vif 2, MMy23-MMy24 for vpx, MMyl2-MMyl4, MMyl2-MMyl3bis for nef2.

It should be noted that thanks to their arrangement on the genome, the primers used for the amplification can be combined in such a way that they can be used as a probe, either after labeling with 32p by kination, or for use in the technique of cold probes to verify the specificity of the amplification band observed during a "Southern transfer" analysis. In addition to the classic combination of primers so that a 3rd oligonucleotide can serve as a specific internal probe, it should be noted the special case of the genes vifl / vpr and vif2 / vpx due to the overlap of these genes, which allows cross-detection . In addition, during a sequencing analysis of the amplified DNA, these oligonucleotides can be used as specific primer for the
DNA polymerase allowing a double sequencing in each direction, therefore a double reading of the sequences, thus removing any ambiguities of interpretation.

 The subject of the invention is also the primers, as defined above, labeled, in particular in a radioactive or enzymatic manner, as well as their use as nucleotide probes, in particular within the framework of the in vitro diagnostic method as described above.

The primers of the invention can also be used for the implementation of a method of in vitro diagnosis of the infection of monkeys (macaque, mangabeys monkey or green monkey) by the virus of the type
SIV, this method repeating the main characteristics of that described above.

 The invention also relates to diagnostic kits for implementing the above-mentioned in vitro diagnostic methods. By way of example, a diagnostic kit of the present invention comprises - at least one pair of oligonucleotide primers according to the invention, each pair comprising a primer hybridizing to one of the strands of the nucleic acid sequence to detect, and a primer hybridizing with the complementary strand of the latter under the conditions defined above, - reagents suitable for carrying out the cycle of amplification operations, in particular DNA polymerase, and four different triphosphate nucleotides, and the reaction medium called "10 X buffer" described above.

- one (or more) labeled probe capable of hybridizing with the amplified nucleic acid sequence (s) to be detected.

 The invention also relates to the use of the primers indicated above for the implementation of a process for the synthesis of proteins encoded by the amplified nucleic sequences.

Another subject of the invention is the use of antisense oligonucleotide primers as antiviral agents, in particular in the fight against
SIDA, as well as pharmaceutical compositions containing these antisense primers in association with a pharmaceutically acceptable vehicle.

 The invention also relates to the immunogenic compositions consisting of translation products of the nucleotide sequences according to the invention and the translation products of the nucleotide sequences amplified from the primers defined according to the invention.

Claims (20)

 1. Nucleotide sequence characterized in that its sequence - either is chosen from those which are contained in one of the nucleic sequences included in the gag and vpr genes of the HIV-1, HIV-2 and SIV viruses, or in the nef2 genes , vif2 and vpx of HIV-2 viruses, and SIV, or in the env, nefl and vifl genes of HIV-1 viruses, - either (in particular for the longest primers) contains one of the above nucleic sequences derived from HIV -1 or HIV-2 or SIV, or contains a nucleic sequence complementary to one of these latter sequences, it being understood that any additional nucleotides which "extend" the nucleic sequence of the genus in question, on the 3 ′ end side or 5 ′, preferably coincide with those which are placed below and at the 3 ′ or 5 ′ ends corresponding within the complete sequence of the viruses of the HIV-1, HIV-2 or SIV type, mentioned above, - either, if the sequence of this primer is not identical & one of the abovementioned nucleic sequences, or is not complementary to one of these sequences, is nevertheless capable of hybridizing with a nucleic sequence derived from HIV-1 viruses, and / or with a nucleic sequence derived from HIV-2 or SIV virus mentioned above.  2. Nucleotide sequence according to claim 1, characterized in that it is included in the genes of HIV-1 Bru virus, HIV-1 Mal, HIV-1 Eli, and / or HIV-2 ROD virus, and / or SIV MAC viruses  3. Sequences according to claim 1 or claim 2, contained in the gag gene of viruses HIV-1 Bru, HIV-1 Mal, HIV-1 ELi, HIV-2 ROD and SIV-MAC, these sequences being characterized by the following nucleotide sequences MMyl: TGG CGC CCG AAC AGG GAC  or T  : S, 636-653, 635-652, 636, 653, 859-876, 834-851 MMy2: GGC CAG GGG GAA AGA AAA A  : S, 854-872,864-888,848-872,1160-1184, 1124-1148 MMy3: TGC CCA TAC AAA ATG TTT TA  or C T T AS, 900-881,916-897,900-881,1212-1193,1176-1157 MMy4: TGC ATG GCT GCT TGA TG AS, 1385-1369,1419-1403,1385-1369,1703-1687,1667-1651  4. Sequences according to claim 1 or claim 2 contained in the vpr gene of viruses HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV-MAC, these sequences being characterized by the following nucleotide sequences MMyl8: GAT AGA TGG AAC AAG CCC CAG  : S, MMyl9: TCC ATT TCT TGC TCT CCT CTG T  : AS.  5. Sequences according to claim 1 or claim 2, contained in the nef2 gene of viruses HIV-2 ROD, and SIV-MAC, these sequences being characterized by the following nucleotide sequences MMyl2: AGA GAC TCT TGC GGG CGC GTG  : S, 9165-9185, MMyl3: ATA TAC TTA GAA AAG GAA GAA GG  : S, 9542-9564, MMyl3bis: CCT TCT TCC TTT TCT AAG TAT  AS, 9564-9542, MMyl4: AGC TGA GAC AGC AGG GAC TTT CCA  AS, 9956-9933,  6. Sequences according to claim 1 or claim 2 contained in the vif2 gene of viruses HIV-2 ROD and SIV-MAC, these sequences being characterized by the following nucleotide sequences MMy20: TAT GGA GGA GGA AAA GAG ATG GAT AGT S, MMy21: TAG GAC TTA TTT CCC TTG CTT T  : S, MMy2lbis: AAA GCA AGG GAA ATA AGT GCT A  : AS, MMy22: CCC TTG TTC ATC ATG CCA GTA T  : AS,  7. Sequences according to claim 1 or claim 2 contained in the env gene of viruses HIV-1 Bru, HIV-1 Mal and HIV-1 Eli, these sequences being characterized by the following nucleotide sequences MMyS: CCA ATT CCC ATA CAT TAT TGT GCC CC: S, 6905-6930,6903-6928,6860-6885 MMy6: AAT GGC AGT CTA GCA GAA GAA GA  : S, 7055-7077,7053-7075,7010-7032 MMy7: ATC CTC AGG AGG GGA CCC AGA AAT T  : S, 7360-7384, 7349-7373,7306-7330 MMy7bis: AAT TTC TGG GTC CCC TCC TGA GGA T  : AS, 7384-7360,7373-7349,7330-7306 MMy8: GTG CTT CCT GCT GCT CCC AAG AAC CC  : AS, 7857-7832,7846-7821,7800-7775  8. Sequences according to claim 1 or claim 2 contained in the nefl gene of viruses HIV-1 Bru, HIV-1 Mal and HIV-1 Eli, these sequences being characterized by the following nucleotide sequences MMy9: ATG GGT GGC AAG TGG TCA AAA AGT AG  or A S, 8844-8869 MMyl0: AAA AGA AAA GGG GGG ACT GGA  : S, 9116-9136, MMylObis: TCC AGT CCC CCC TTT TCT TTT  : S, 9136-9116 MMyll: AAA GTC CCC AGC GGA AAG TCC C  : AS, 9503-9483,  9. Sequences according to claim 1 or claim 2 contained in the vifl gene of the HIV-1 Bru, HIV-1 Mal and HIV-1 Eli viruses, these sequences being characterized by the following nucleotide sequences MMyl5: GAT TAT GGA AAA CAG ATG GCA GGT GAT S, MMyl6: GCA GAC CAA CTA ATT CAT CTG TA S, MMyl6bis: TAC AGA TGA ATT AGT TGG TCT GC  : AS, MMyl7: CTT AAG CTC CTC TAA AAG CTC TA  : AS,  10. Method for gene amplification of nucleic sequences of viruses of the HIV-1 and / or HIV-2 type, and / or SIV, carried out from a biological sample, this process mainly comprising the following steps: - a step of extraction of the nucleic acid to be detected belonging to the genome of the HIV-1 type virus HIV-2, or SIV possibly present in the aforementioned biological sample, and, where appropriate, a step of treatment using a reverse transcriptase of said nucleic acid if the latter is in the form of 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 one of claims 1 to 9, by bringing the above-mentioned strands into contact with at least one pair of aforementioned primers  formation from primers of DNA complementary to the strands on which they are hybridized in the presence of a DNA polymerase and four different nucleoside triphosphate (dNTP), which leads to the formation of a greater number of double-stranded nucleic acids å to detect that & the previous denaturation step, this cycle being repeated a determined number of times to obtain said nucleic sequence å to detect possibly present in the biological sample in a sufficient proportion to allow its detection, - a detection step of the possible presence of the nucleic acid belonging to the genome of the virus of the HIV-1 and / or HIV-2 and / or SIV type in the biological sample.  11. Method according to claim 10, characterized in that the hybridization step is carried out at 60 ° C for 1 minute 30 seconds in the buffer described in claim 1.  12. Method according to claim 10, characterized in that the step of extracting the viral DNA comprises the following steps suspension of the cell pellet in 0.5 ml of pyrolized water in a large piston Potter, grinding of said cells by "round trip", addition of Triton X100 for 1 final concentration of 0.1%, heat denaturation for 15 minutes at 100 μl, centrifugation to remove cellular debris,. DNA precipitation overnight at -20 ° C by addition of 2.5 volumes of absolute ethanol and 10% of the final volume of 1 Molar sodium acetate.  13. Method according to claim 10, characterized in that the step of reverse transcription of the viral RNA comprises the following steps - 10 Hg of extracted RNA resuspended in water is placed in the presence of the pair of primers at a concentration of 0.8 pM each, in a final volume of 40 1, the whole is denatured at 100in for 10 minutes then immersed in ice water, - 10 l of the following mixture are added: 5 l buffer "10 X buffer" (comprising: Tris-HCl, pH = .8.9 50 mM; (NH4) 2 SO4; 15 mM; MgCl2, 5 mM; ss-mercapto-ethanol: 10 mM; gelatin: 0, 25 mg / ml) + 1 unit of reverse transcriptase + 1 unit of Taqpolymerase + 1 l of the mixture of 4 dNTPs at 25 mM each + QSP water 10 p1, the manufacture of the cDNA is done by the action of reverse transcriptase at 42iC for 13 minutes, then heating at 95 C for 3 minutes to destroy the reverse transcriptase.  14. -Application of the method according to any one of claims 9 to 13 or in vitro diagnosis of the infection of an individual by a virus of the type HIV-1 and / or HIV-2, or of an animal with at least one of the three viruses (HIV-1, HIV-2, SIV).  15. Application of the method according to any one of claims 9 to 13 & the amplification of nucleotide sequences of the genomes of viruses of the HIV or SIV type, followed by the translation of these amplified sequences, from the nucleotide primers according to the one of claims 1 to 9, in proteins.  16. Immunogenic compositions comprising one (or more) translation product of the nucleic sequences according to one of claims 1 to 9, and / or one (or more) translation product of the nucleotide sequences amplified by the method according to one of claims 9 to 13  17. Amplification buffer for implementing the amplification cycle of a method according to any one of claims 9 to 13, characterized in that it consists of Tris-HCl, pH 8.9: 50 mM (NH4) 2 SOx: 15 mM; MgC12: 5 mM, B-mercapto-ethanol 10 mM; gelatin: 0.25 mg / ml.  18. Oligonucleotide primer couples, for the implementation of a method according to one of claims 9 & 13, following MMyl-MMy4 (amplified fragment: 650 bases), MMy2-MMy4 (538 bases), MMyl-MMy3 (265 bases), MXyl8-MMyl9 (275 bases), MMy5-MMy8 (953 bases), MMy6-MMy8 (803 bases) , MMy7-MMy8 (498 bases), MMyS-MMy7bis (480 bases), MMy6 NMy7bis (330 bases), MMy9-MMyll (660 bases), MMy9 MMylObis (292 bases), MMyl5-MMyl7 (600 bases), MMyl5 MMyl6bis (335 bases), MMyl6-MMyl7 (290 bases), MMy20 MMy22 (655 bases), MMy2O-MMy2lbis (350 bases), MMy21- MMy22 (325 bases), MMy23-MMy24 (230 bases), MMyl2-MMyl4 (791 bases), MMyl2-MMyl3bis (600 bases).  19. Kit for implementing a method according to one of claims 9 d 13 comprising - at least one pair of oligonucleotide primers according to any one of claims 1 to 9 or according to claim 18, - reagents suitable for carrying out the amplification cycle, in particular DNA polymerase, and four different nucleotide triphosphate, and the reaction medium according to claim 17, - one (or more) labeled probe capable of hybridizing with the amplified nucleic acid sequence (s) to be detected.  20. Composition for the treatment of viral diseases, in particular AIDS, comprising at least one nucleotide sequence according to one of claims 1 to 9 in association with a pharmaceutically acceptable vehicle.