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

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 HIV type or of monkey immunodeficiency retrovirus of SIV type.

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 (HIV-1) and No. 87 / 400.151.4 and EP 0269520 (HIV-2). These retroviruses have been isolated from several patients with symptoms of lymphadenopathy or Acquired Immunodeficiency 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 regard to these lymphocytes when they multiply therein, thus causing between others generalized and persistent polyadenopathies, or AIDS.

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

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

Continued study of the HIV-1 and HIV-2 retroviruses 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 1-522, under the reference name LAV-2 ROD.

Similarly, 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 what follows by the expression HIV.

Diagnostic methods <u style="single">in vitro</u> infections with viruses of the HIV-1 or HIV-2 type currently existing, call for the detection of ANTI-HIV-1 or anti-HIV-2 antibodies possibly present in a biological sample (biopsy) or in a biological fluid , for example 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, in particular in the case of a recent infection of an individual with HIV-type viruses.

Gene amplification techniques are a considerable supplement to the development of diagnostic methods <u style="single">in vitro</u> particularly susceptible to viral diseases. Among these gene amplification techniques, mention may be made of the PCR (Polymerase Chain Reaction) technique as described in European patent applications No. 86 / 302.298.4 of 03/27/1986 and No. 87 / 300.203.4 of 01/09/1987, or the so-called "Qβreplicase" technique described in Biotechnology, vol.6, page 1197 (October 1988) and that using an RNA polymerase (T7RNA polymerase) described in the patent application International No. WO89 / 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. Indeed, 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 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 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, inter alia, the amplification of the genome of all viruses of the HIV and SIV type, with yields considered to be maximum in the current state of the art and above all avoiding the presence of numerous non-specific bands .

The primers used in 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 the use of oligonucleotide primers, of around 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 uses any nucleotide sequence characterized in that its sequence:<ul id="ul0001" list-style="dash" compact="compact"><li>either is chosen from those which are contained in one of the nucleotide sequences included in the gag gene of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV MAC viruses, and more particularly from those which are contained in the nucleotide sequences defined below,</li><li>either (in particular for the longest sequences) contains one of the abovementioned nucleotide sequences derived from HIV-1 Bru, or HIV-1 Mal, or HIV-1 Eli or HIV-2 ROD or SIVMac, or contains a complementary nucleotide sequence of one of these latter sequences, it being understood that any additional nucleotides which "extend" over the nucleotide sequence of the genus in question, on the 3 ′ or 5 ′ end side, preferably coincide with those located below the 5 'ends</li></ul>or 3 ′ corresponding within the complete sequence of the viruses of the HIV-1, HIV-2 or SIV MAC type, mentioned above,<ul id="ul0002" list-style="dash" compact="compact"><li>either, if this nucleotide sequence is not identical to one of the above nucleotide sequences, or is not complementary to one of these sequences, is nevertheless capable of hybridizing with a nucleotide sequence derived from HIV- 1 Bru, HIV-1 Mal, HIV-1 Eli, and / or with a nucleotide sequence derived from the HIV-2 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), recommended for optimum performance.</li></ul>

The numbering of the nucleotides mentioned below 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 in European patent application No. EP 0 253 701.

The sequences of the invention are synthesized on a synthesizer marketed by Applied Biosystems (phosphoro-amidites method, or on any other device using a similar method.

The invention relates more particularly to the use of 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, c ' that is to say if the oligonucleotide is oriented respectively in the direction 5'o → 3 'or in the direction 3' o → 5 '):<ol id="ol0001" compact="compact"><li>1) sequences common to the genomes of the HIV-1, HIV-2 and SIV viruses (the series of numbers spaced apart by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-1 Bru, HIV-1 Mal viruses , HIV-1 Eli, HIV-2 ROD and SIV):<ul id="ul0003" list-style="bullet" compact="compact"><li>specific sequences of the gag gene of the genome of the above-mentioned viruses (gene coding for a group of antigens specific to the nucleoide of these viruses).</li></ul></li></ol>

Certain variants can be made at certain positions of the nucleotide sequences indicated below, without the hybridization properties of these nucleotide sequences with the genes of viruses of the HIV and / or SIV type being affected. The nucleotide sequences comprising these variants are shown below the initial nucleotide sequences from which they are derived by replacement of one or more bases. The bases modified with respect to those of the initial nucleotide sequences are indicated in any letter vertically from the positions corresponding to the bases which have been replaced in these initial sequences; while the bases of the initial sequences which have not been replaced in the sequences comprising these variants are indicated using dotted lines.

The synthesis of the primers is done using all the variants simultaneously. It is the mixture of all the variants for a given sequence which is used in the tests.<img file="EP0806484B1_D0001.tif" /><img file="EP0806484B1_D0002.tif" /><img file="EP0806484B1_D0003.tif" /><img file="EP0806484B1_D0004.tif" /><img file="EP0806484B1_D0005.tif" /><img file="EP0806484B1_D0006.tif" /><img file="EP0806484B1_D0007.tif" /><img file="EP0806484B1_D0008.tif" />

A subject of the invention is also the use in the method of synthesis of the sequences (or primers) having a nucleotide structure complementary to those of the primers defined above.

It also relates to the use in the method of synthesis of the nucleotide sequences having certain mutations compared to those defined above without the hybridization properties, as defined above, of these sequences being modified. The percentage of nucleotides different from those constituting the sequences described above, without however affecting the hybridization properties of the sequences of the invention, can reach 40%.

In general, in the case of a primer (S), a larger number of mutations will be tolerated on the 5 'side than on the 3' side of the primer, the 3 'side in front of' hybridize perfectly with a specific strand of a nucleic sequence to allow the amplification of this sequence. In the case of an anti-sense primer (AS), tolerance is allowed on the 3 'side.

The primers as defined above and can comprise a conservation 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 used in the invention is to give a clear amplification band, generally devoid of non-specific bands when the technical indications for use described in the present invention are used. 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 the use of 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 RNA as described in European patent application n ° 88 / 307.102.9 dated 08/01/1988.

A subject of the invention is in particular the use of the primers described above for the implementation of a method of gene amplification of nucleic sequences of viruses of the HIV-1 and / or HIV-2 type, and / or The gene amplification process mainly includes the following steps:<ul id="ul0004" list-style="dash" compact="compact"><li>a step of extracting the nucleic acid to be detected belonging to the genome of the virus of the HIV-1, HIV-2 or SIV type and, if appropriate, a step of treatment 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 (this last step being further designated below by step of reverse transcription of the viral RNA),</li><li>a cycle comprising the following stages:<ul id="ul0005" list-style="bullet" compact="compact"><li>denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid,</li><li>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 below,</li><li>formation from DNA primers complementary to the strands on which they are hybridized in the presence of a polymerization agent (DNA polymerase) and four different nucleoside triphosphate (dNTP), which leads to the formation of a larger number of double stranded nucleic acids to be detected 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,</li></ul></li></ul>

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

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 organism, especially inside the blood cells, its RNA is copied into DNA by a 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 method with chloroform phenol - presents the following steps:<ul id="ul0006" list-style="bullet" compact="compact"><li>suspension of the cell pellet in 0.5 ml of pyrolized water in a Potter large piston,</li><li>shredding of cells known as "round trip",</li><li>addition of Triton X100 for 1 final concentration of 0.1%,</li><li>heat denaturation for 15 to 25 minutes at 100 ° C,</li><li>short centrifugation to remove only cellular debris,</li><li>DNA precipitation overnight at -20 ° C by adding 2.5 volumes of absolute ethanol and 10% of the final volume of 3 Molar sodium acetate. The DNA is then recovered and then resuspended in pyrolyzed water after having been washed twice with ethanol at 70 °. It should be noted that this method allows the joint precipitation of DNA and RNA, which authorizes the detection of the genomic message of viruses of HIV or SIV types by using the method called "direct DNA PCR" or by that called "PCR -RNA ".</li></ul>

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 be carried out when the diagnosis <u style="single">in vitro</u> of the invention is produced from biological samples containing viruses of the HIV-1 and / or HIV-2 and / or SIV type, the genomes of which 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 viral RNA reverse transcription step is carried out as follows:<ul id="ul0007" list-style="dash" compact="compact"><li>10 <i>µ</i>g of RNA extracted resuspended in water is brought into contact with the pair of primers at a concentration of 40 <i>µ</i>M each, in a final volume of 40 <i>µ</i>l. The whole is denatured at 100 ° C for 10 minutes then immersed in ice water,</li><li>we add 10 <i>µ</i>l of the following mixture: 5 <i>µ</i>l of the "10 X buffer" buffer described below + 1 unit of reverse transcriptase (from AMV (Avian Myeloblastosis Virus) or MuMLV (Moloney Leukemia Virus)) + 1 unit of Taq-polymerase + 1 <i>µ</i>l the mixture of 4 dNTPs at 25 mM each + QSP 10 water <i>µ</i>l. The final volume is therefore 50<i>µ</i>l.</li></ul>

This reaction takes place in two stages:<ul id="ul0008" list-style="dash" compact="compact"><li>a) 1st step: manufacture of the cDNA by the action of reverse transcriptase at 42 ° C for 13 minutes,</li><li>b) 2nd step: conventional 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.</li></ul>

The denaturation step is carried out in the presence of the pair (s) of primers (or primers) of the invention. Indeed, as has been specified above, one of the characteristics of the oligonucleotides (or primers) of the invention is to give a clear amplification band, generally devoid of non-specific bands, when they are used under the conditions that follow :<ul id="ul0009" list-style="dash" compact="compact"><li><u style="single">hybridization</u> : the primers (1<i>µ</i>l of a solution at 40 <i>µ</i>molar (40 <i>µ</i>M) of each primer) are placed in the presence of the template DNA (100 to 300 ng) for the first denaturation-reassociation step; the mixture is heated for 10 minutes at 100 ° C. and then the tubes containing this mixture of DNA-matrix and primers are immersed in water containing ice in order to increase the rate of DNA-matrix / primers reassociation. Primers should be used at a final concentration in the next amplification step of 0.8<i>µ</i>M each.</li><li><u style="single">amplification:</u> the 4 A dNTP is added to the preceding medium, each being used at 0.5 <i>µ</i>molar in final solution (50 <i>µ</i>l), and a unit of Taq-polymerase for a reaction medium of 50 <i>µ</i>this step is carried out in an amplification buffer of the present invention, generally designated under the name of "10 X buffer", the composition (when diluted 1/10 °) is as follows: Tris-HCl , pH 8.9: 50 mM; (NH₄)₂ SO₄ : 15 mM; MgCl2: 5 mM;<i>β</i>-mercapto-ethanol: 10 mM; gelatin: 0.25 mg / ml. We add 5<i>µ</i>l of this buffer and water qs 50 <i>µ</i>l in the previous middle.</li></ul>

The amplification cycles are carried out as follows: 30 to 40 cycles composed of:<ul id="ul0010" list-style="bullet" compact="compact"><li>94 ° C for 10 seconds (denaturation),</li><li>60 ° C for 1 minute 30 (hybridization),</li><li>78 ° C for 1 minute 30 (elongation).</li></ul>

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

The accuracy of the temperatures indicated to within ± 0.3 ° C, as well as their stability during the various cycles, represent essential conditions for obtaining maximum yields as well as the absence of non-specific bands.

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 goes without saying that the above conditions represent optimal conditions for a final reaction medium of 50 <i>µ</i>1, and that these conditions can be modified as a function of the final volume of the reaction medium.

The polymerization agent used in the elongation stage of the cycle is a thermostable DNA polymerase, in particular Taq polymerase, the amplifiose from the firm Appligène or any thermostable DNA polymerase that can be marketed.

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

The pairs of primers, which can be used, for example, for the gene amplification method are the following:<ul id="ul0011" list-style="dash" compact="compact"><li>MMy1-MMy4, MMy2-MMy4, MMy1-MMy3, MMy4bis-MMy28bis for the gag gene,</li></ul>

However, the combinations between primers "S" and "AS" described above are not limiting and can be varied as desired by the user.

The sizes of the nucleotide fragments synthesized using the pairs of primers mentioned above as examples, are indicated in the following table:<ul id="ul0012" list-style="none" compact="compact"><li>(the figures indicated in the table below represent the number of nucleotides of the fragments synthesized, and the "dashes" indicate that the pairs of primers tested do not make it possible to characterize the corresponding viral strains).</li></ul><tables id="tabl0001" num="0001"><table frame="all"><title>Table I</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52mm" colsep="1" /><colspec colnum="2" colname="col2" colwidth="11mm" colsep="1" /><colspec colnum="3" colname="col3" colwidth="11mm" colsep="1" /><colspec colnum="4" colname="col4" colwidth="11mm" colsep="1" /><colspec colnum="5" colname="col5" colwidth="11mm" colsep="1" /><thead><row><entry namest="col1" nameend="col3" colsep="1" rowsep="1" align="center" valign="top">gag</entry><entry namest="col4" nameend="col5" colsep="1" rowsep="1" align="center" valign="top">gag</entry></row><row><entry namest="col1" nameend="col5" colsep="1" rowsep="1" align="center" valign="top">: MMy1-MMy3: MMy1-MMy4: MMy2-MMy4: MMy4bis-MMy28bis:</entry></row></thead><tbody><row><entry namest="col1" nameend="col1" align="left" valign="top">HIV1-BRU:</entry><entry namest="col2" nameend="col2" align="center" valign="top">265</entry><entry namest="col3" nameend="col3" align="center" valign="top">750</entry><entry namest="col4" nameend="col4" align="center" valign="top">532</entry><entry namest="col5" nameend="col5" align="center" valign="top">671</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HIV1-MAL:</entry><entry namest="col2" nameend="col2" align="center" valign="top">282</entry><entry namest="col3" nameend="col3" align="center" valign="top">785</entry><entry namest="col4" nameend="col4" align="center" valign="top">556</entry><entry namest="col5" nameend="col5" align="center" valign="top">671</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HIV1-ELI:</entry><entry namest="col2" nameend="col2" align="center" valign="top">265</entry><entry namest="col3" nameend="col3" align="center" valign="top">750</entry><entry namest="col4" nameend="col4" align="center" valign="top">538</entry><entry namest="col5" nameend="col5" align="center" valign="top">674</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">HIV2-ROD:</entry><entry namest="col2" nameend="col2" align="center" valign="top">354</entry><entry namest="col3" nameend="col3" align="center" valign="top">845</entry><entry namest="col4" nameend="col4" align="center" valign="top">544</entry><entry namest="col5" nameend="col5" align="center" valign="top">663</entry></row><row><entry namest="col1" nameend="col1" align="left" valign="top">IF V :</entry><entry namest="col2" nameend="col2" align="center" valign="top">343</entry><entry namest="col3" nameend="col3" align="center" valign="top">844</entry><entry namest="col4" nameend="col4" align="center" valign="top">544</entry><entry namest="col5" nameend="col5" align="center" valign="top">668</entry></row></tbody></tgroup></table></tables>

The invention also relates to the use of oligonucleotides as described above and comprising sugars in a conformation. Such oligonucleotides have the characteristic of reversing the direction of the double helix formed with the matrix (strand of the virus genome), this double helix thus passing from the "S" state to the "AS" state.

The invention also relates to the use of the oligonucleotides described above, certain nucleotides of which are methylated and / or contain one or more sulfur atoms, in particular on the adenines. Such oligonucleotides have the characteristic of increasing the stability of the double helix, and consequently of better hybridizing with the DNA strand to be amplified.

The invention also relates to the use of oligonucleotides as described above and in the form known as "modified bases" comprising nucleotides on which are covalently grafted chromophoric agents (flat aromatic molecules such as acridine orange), in particular according to the method described in the article by C. Hélène published in "la Vie des Sciences", reports, general series, volume 4, n ° 1, p. 17-37. Such oligonucleotides have the characteristic of being easily detectable, in particular by fluorescence.

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

The process for the synthesis of 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:<ul id="ul0013" list-style="none" compact="compact"><li>at. synthesis of the nucleotide sequence by a method of gene amplification of nucleotide sequences of HIV-1, H IV-2 or SIV type viruses, using at least two oligonucleotide primers, the sequences of which each consist of<ul id="ul0014" list-style="none" compact="compact"><li>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 HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 Rod and SIV Mac viruses, or</li><li>ii. a sequence complementary to a sequence as defined in i .; and</li></ul></li><li>b. recovery of the nucleotide sequence thus amplified and translation into protein.</li></ul>

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

Another specific synthetic 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 relative to the sequence of the gag gene of an HIV-1 Bru virus , HIV-1 Mal, HIV-1 Eli, or HIV-2 ROD or SIV Mac. and keep the hybridization properties with the genomes of 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 the gene amplification method comprises the following steps:<ul id="ul0015" list-style="none" compact="compact"><li>at. a step of extracting the nucleic acid belonging to the genome of the virus of HIV-1, HIV-2 or SIV type, and optionally a step of treatment using a reverse transcriptase of said nucleic acid if the latter is under form of RNA.</li><li>b. a cycle comprising the following stages:<ul id="ul0016" list-style="none" compact="compact"><li>i. denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid,</li><li>ii. hybridization of each of the nucleic acid strands, obtained during the preceding denaturation step, with at least one nucleotide primer, by bringing the above-mentioned strands into contact with at least one pair of primers according to one of claims 1 or 2,</li><li>iii. formation, from the primers, of DNAs complementary to the strands on which said primers are hybridized in the presence of a DNA polymerase and of four different nucleoside triphosphates (dNTP), which leads to the formation of a greater number of acids double-stranded nucleic acids than in the previous denaturation step,</li></ul></li></ul>this cycle being repeated a number of times determined to obtain said nucleotide 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<ul id="ul0017" list-style="none" compact="compact"><li>i. at least one sequence chosen from the following sense sequence group:<img file="EP0806484B1_D0009.tif" />and at least one sequence chosen from the following group of antisense sequences<img file="EP0806484B1_D0010.tif" /></li><li>ii. a sequence complementary to a sequence as defined in i .; or</li><li>iii. a sequence having at least 60% identity with one of the sequences defined in i or ii and capable of hybridizing at a temperature of 60 ° C ± 1 ° C with the genomes of the HIV-1 Bru, HIV-1 viruses Mal, HIV-1 Eli, HIV-2 ROD and SIV Mac.</li></ul>

Another method according to the invention is characterized in that at least two of the following primers mixtures are used for the amplification:<img file="EP0806484B1_D0011.tif" /><img file="EP0806484B1_D0012.tif" /><img file="EP0806484B1_D0013.tif" /><img file="EP0806484B1_D0014.tif" /><img file="EP0806484B1_D0015.tif" /><img file="EP0806484B1_D0016.tif" /><img file="EP0806484B1_D0017.tif" /><img file="EP0806484B1_D0018.tif" /><img file="EP0806484B1_D0019.tif" />

Another process according to the invention is characterized in that it is carried out under the following conditions:<ul id="ul0018" list-style="dash" compact="compact"><li>for the hybridization step: 1 μl of a 40 μmolar solution of each primer is brought into contact with 100 to 300 ng of template DNA for the first denaturation-reassociation step; the mixture is heated for 10 minutes at 100 ° C. and then the tubes containing this mixture of DNA-template and primers are immersed in water containing ice, the primers being used at a final concentration in step d amplification which follows by 0.8 μM each;</li><li>for the amplification step, the 4 dNTPs are added to the preceding medium, each being used at 0.5 μmolar in 50 μl of final solution, and one unit of Taq-polymerase for a reaction medium of 50 μl, this step being carried out in the amplification buffer designated under the name of "10 X buffer" comprising, when diluted 1/10 in the final solution: Tris-HCl, pH = 8.9: 50 mM; (NH₄)₂ SO₄ ; 15 mM; MgCl₂ ; 5 mM; β-mercapto-ethanol: 10 mM; gelatin: 0.25 mg / ml.</li></ul>

Another method according to the invention is characterized in that the translation step is carried out by transformation of appropriate host cells using vectors containing said amplified sequences and recovery of 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 primers mixtures:<ul id="ul0019" list-style="none" compact="compact"><li>at. MMy1-MMy4<img file="EP0806484B1_D0020.tif" /><img file="EP0806484B1_D0021.tif" /></li><li>b. MMy2-MMy4<img file="EP0806484B1_D0022.tif" /><img file="EP0806484B1_D0023.tif" /></li><li>vs. MMy1-MMy3<img file="EP0806484B1_D0024.tif" /><img file="EP0806484B1_D0025.tif" /></li><li>d MMy4Bbis-MMy28bis<img file="EP0806484B1_D0026.tif" /><img file="EP0806484B1_D0027.tif" /></li></ul>

The last translation step is carried out in particular by transformation of appropriate host cells using vectors containing said amplified sequences, and recovery of the proteins produced in these host cells.

Also concerned are the polypeptides resulting from the translation of the nucleotide sequences (or primers) of the invention.

The present application also describes the immunogenic compositions comprising one or more translation products of the nucleotide sequences according to the invention, and / or one or more translation products of the nucleotide sequences amplified according to the methods described above from the primers defined according to l invention, these translation products being associated with a pharmaceutically acceptable vehicle.

The present application describes the 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 one or more products of translation 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:<ul id="ul0020" list-style="dash" compact="compact"><li>incubation of genomic DNA, isolated from one of the above-mentioned HIV or SIV type viruses, with DNAase I, then addition of EDTA and purification by extraction with a phenol / chloroform / isoamyl alcohol mixture (25 / 24/1) then with ether,</li><li>processing of the DNA thus extracted with<u style="single">Eco</u> R1 methylase in the presence of DTT, and purification by extraction as described above,</li><li>incubation of the DNA thus purified with the 4 deoxynucleotide triphosphates dATP, dCTP, dGTP, and dTTP in the presence of T4 DNA polymerase and DNA ligase <u style="single">E. coli,</u> then purification according to the method described above,</li><li>the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid sought using a suitable probe.</li></ul>

A particularly advantageous process for preparing the nucleotide sequences of the invention comprises the following steps:<ul id="ul0021" list-style="dash" compact="compact"><li>DNA synthesis using the automated β-cyanethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325 (1986),</li><li>cloning the nucleic acids thus obtained into an appropriate vector and recovering the nucleic acid by hybridization with an appropriate probe.</li></ul>

Another method for preparing the nucleotide sequences of the invention comprises the following steps:<ul id="ul0022" list-style="dash" compact="compact"><li>the assembly of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are 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),</li><li>cloning the nucleic acids thus obtained into an appropriate vector and recovering the desired nucleic acid by hybridization with an appropriate probe.</li></ul>