Nucleotides sequences derived from the genome of type hiv-1,hiv-2 and siv retroviruses, and their applications especially for the amplification the genomes of these retroviruses and for in vitro diagnosis of infections due to these viruses

W 90/15066 ~ 0 6 ~ ~ ~ PCT / FR90 / 00393 1 PdDCLEOTIDIQIIES SEQUENCES FROM GE241OK $ OF RETROVIRUSES OF THE HI -l, RIV-2 AND SIV TYPE, AND THEIR APPLICATIONS NOTA.MMElNT FOR; THE AMPLIFICATIC2d OF THE GENOMES OF THESE RETROVIRUSES AND FOR DIAGNOSIS INVITRO OF IAïIrECTI 2dS DUE TO THESE VIRUSES.

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

The invention relates in particular to the application of these sequences to in vitro diagnostic methods 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 together 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 showing symptoms of lymphadenopathy or Acquired Immunodeficiency Syndrome (AIDS).

The retroviruses of the HIV-2 type, like the retroviruses of the HIV-1 type, are characterized by a tropism for human T4 lymphocytes and by a cytopathogenic effect with regard to these lymphocytes when they multiply there, to then cause between others generalized and persistent polyadenopathies, or AIDS.

Another retrovirus, called SIV-1, this name; nation replacing the previously known name STLV-III, was isolated from the rhesus macaque monkey (Nt.D. DANIEL et al.

Science, 228, 1201 (1985) ~~~ iiL ~~ Dr RE ~ q P- ~ A ~~~ ltE W + o 90/15066 PCI '/ FR90 / 00393 2 NL LETWIN et al., Science, 230, 71 ( 1985) under the name "STLV-Illmac").

Another retrovirus, designated "STLV-IIIAGN" (or SIVAGM) was isolated from wild green monkeys.

But unlike the viruses present in the rhesus macaque monkey, the presence of STLV-IIIAGm does not appear 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 by the expression SIV (the expression SIV is the abbreviation of "Simian Immunodeficency.

Virus "(Monkey immunodeficiency virus) optionally followed by an abbreviation designating the species of monkey from which they are derived, for example" MAC "for the macaque" or "AGM" for the African green monkey (abbreviation of " African Green Monkey ").

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

The continuation of the study of the HIV-1 and HIV-2 retroviruses has also led to the obtaining of DNA sequences complementary to the RNAs of their genome.

The complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-2 class (iilV-2 ROD) was deposited on 02/21/1986 at the CNCM under the n 'I-522, under the reference name LAV-2 ROD.

Likewise, the complete nucleotide sequence of a cDNA of a retrovirus representative of the HIV-1 class is described by WAIN HOBSON, SONIGO, COLE, DANOS and ALIZON in CE11 (January 1985).

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

In vitro diagnostic methods of infections by viruses of the HIV-1 or HIV-2 FIRE type: The 1% t'O 90 / 1506b 2 8.2.9 PCT / FR30 / 00393 3 currently existing, involve 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 run the risk of being false negative, in particular in the case of a recent infection of an individual with viruses of the HIV type.

Gene amplification techniques are of considerable help in the development of particularly sensitive in vitro diagnostic methods for viral diseases.

Among these gene amplification techniques, mention may be made of the PCR (Polymerase Chain Reaction) technique as described in European patent applications n '86 / 302.298.4 of 03/27/1986 and n 87 / 300.203.4 of 09 / 01/1987, or the so-called “Qpreplicase” technique described in Biotechnology, vol.6, page 1197 (October 1988) and that using an ARDi polymerase (T7RNA polymerase) described in the international patent application No. W089 / 01050.

These techniques make it possible to improve the sensitivity of detection of the nucleic acids of viruses, and require the use of specific synthetic primers.

For the search for viruses of the HIV type, 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 FEUI &: ~~ DE. REPLACEMENT t,, CA 02062829 2006-07-10 4 amplification of certain viral variants of the HIV type. On the other hand, even if a primer is chosen from a conserved region of the genome from one HIV virus to another, its “good functioning” is not for all that guaranteed and can give rise to poor amplification yields.

The present invention provides precisely oligonucleotide primers allowing, among other things, the amplification, in particular for diagnostic purposes, 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 especially avoiding the presence of numerous non-specific bands.

The present invention relates to a method for the gene amplification of gag, pol or vpr sequences of HIV-1, HIV-2 and SIV viruses comprising the use of at least two oligonucleotide primers, of 15 to 30 nucleotides, capable of hybridize to the genome of HIV-1, HIV-2 and SIV at a temperature of 60 C 1 C, chosen from:

(a) the conserved and specific sequences of the gag, pol or vpr genes of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV Mac viruses, and (b) the sequences complementary to the defined sequences in (a).

The present invention also relates to a method of synthesizing a protein or a polypeptide encoded by a polynucleotide obtained by the gene amplification of the genome of HIV-1, HIV-2 or SIV comprising - a step of amplifying the genome of HIV1, HIV-2 or SIV using at least two oligonucleotide primers as defined according to the present invention, CA 02062829 2006-08-10 4a - the introduction of the polynucleotide thus obtained into an appropriate vector, - transformation of host cells using the aforementioned vector, - cultivation of host cells transformed with said vector and recovery of the protein or polypeptide produced by the latter.

The present invention also relates. nucleotide primers, for carrying out the gene amplification of a gene of the genome of HIV-1, HIV-2 or SIV, chosen from the following pairs of primers (a) MMyl-MMy4, (b) MMy2 -MMy4, (c) MMyl-Mmy3, (d) MMy4Bis-MMy28bis, (e) MMyl8-MMy19, (f) MMy29-MMy3Obis, (g) MMy30-MMy3lbis and (h) MMy3l-MMy32bis.

The present invention also relates to the use of at least one pair of nucleotide primers as mentioned above for the in vitro diagnosis of the infection of an individual by HIV-1 or HIV-2 or both.

The present invention also relates to the use of at least two pairs of primers as mentioned above for the cross detection of CA 02062829 2006-08-10 4b several types of viruses of the type HIV-1, HIV-2, SIV or combinations of these.

The present invention also relates to the use of at least two pairs of primers as mentioned above for the. simultaneous detection of several genes of the same virus of the HIV-1, HIV-2 or SIV type.

The present invention also relates to the use of at least one pair of nucleotide primers as mentioned above for the implementation of a method for the gene amplification of nucleic acid sequences of viruses of at least one type chosen from HIV-1. , HIV-2 and. IF V.

The present invention also relates to the use of at least one pair of nucleotide primers as mentioned above for the implementation of a process for the synthesis of a protein or a polypeptide encoded by a nucleic acid belonging to the genome of a virus of the HIV-1, HIV-2 or SIV type and amplified using said at least one pair of primers.

The present invention also relates. a method of in vitro diagnosis of a potential infection of an individual by a virus of the HIV-1 or HIV-2 type or both, or of an animal by at least one of the viruses chosen from HIV-1, HIV-2 and SIV, said method being carried out from a biological sample originating from an individual or an animal under study, and comprising the following steps:

i. a method of gene amplification as mentioned above, carried out on a nucleic acid extracted from said biological sample; and ii. a step of detecting the possible presence of nucleic acid CA 02062829 2006-10-13 4c belonging to the genome of the virus of the HIV-1 or HIV-2 or SIV type or of combinations thereof in the biological sample.

The present invention also relates to a method of in vitro diagnosis of a potential infection of an individual by a virus of the HIV-i or HIV-2 type or both, or of an animal by at least one of the selected viruses. from HzV-1, HIV-2 and SzV, said method being carried out using a biological sample originating from an individual or an animal under study, and comprising the following steps: a:

i. a method of gene amplification as mentioned above, carried out on a nucleic acid extracted from said biological sample; and ii. detecting the possible presence of a product of said amplification process, said product corresponding to a nucleic acid sequence of the genome of the virus type HIV-1 or HXV-2 or SIV or combinations thereof in the biological sample.

The present invention also relates to a kit for implementing a method as mentioned above comprising.

- at least a pair of aligonucleotide primers as mentioned above; and reactions appropriate to the midst of a cycle of amplification operations.

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, CA 02062829 2006-10-13 4d which can be used for the 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 nucleotide sequences included in the gag, vpr and pol genes of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV MAC viruses, or in the nef2, virz and vpx genes of the HIV-2 ROD, and SIV MAC viruses, or in the env, nefL, vit1 and vpr genes of the HIV-1 Bru, HIV-1 Mal, and HIV-1 Eli viruses, and more particularly among those which are contained in the nucleotide sequences defined below, - either (in particular for 1.ee eequences 1es longer) contains one of the aforementioned nucleotide sequences resulting from HxV-1 Bru, or HIV-1 Mal, or HIV-1 WO 90/15066 PCT / FR90 / 00393 2062829 Eli or HIV-2 ROD or SIVMac, or contains a nucleotide sequence complementary to one of the latter sequences, it being understood that the possible additional nucleotides which "overlap" the nucleotide sequence of the genus in question, on the side of the 3 'or 5' ends , preferably coincide with those which are placed on this side of the 5 'or 3' ends corresponding to the same complete sequence of viruses of the HIV-1, HIV-2 or SIV MAC type, mentioned above, - either, if this nucleotide sequence is not identical to one of the aforementioned nucleotide sequences., or is not complementary to one of these sequences, is nevertheless capable of hybridizing with a nucleotide sequence derived from the HIV-1 Bru viruses , 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 yield.

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 E1i viruses have been described by MONTAGNIER, SONIGO, WAIN-HOBSON and ALIZON in European patent application No. 86.401380 of 06/23/86).

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 oligonucleotide sequences characterized by the following nucleotide sequences (represented ~ EUIL '~ Dt- REMPLA ~ EME _, ... , WO 90/15066 2062829 PCT / FR90 / 00393 6 in the 5 '- 3' direction; the initials reS, r and "AS" indicate whether the oligonucleotide is sense or antisense, that is to say if the oligonucleotide is oriented respectively in the 5'o-- 3 'direction or in the 3' o direction ~ 5 ') 1') sequences common to the genomes of the HIV-1, HIV-2 and SIV viruses (the series of numbers spaced by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-1 Bru and HIV viruses -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 specific antigens of the nucleoid of these viruses).

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 represented 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 full letters vertically at 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 with 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 that is used in the tests.

E'E U baL ~. DE RErO P LACW E Pe. 1M WO 90/15066 2 ~ ~ ~ ~ ~ ~ PCT / F1290 / 00393 7 Mriyl TGG CGC CCG AAC AGG GAC ... ... T. ... ... ...

S, 636-653, 635-652, 636-653, 859-876, 834-851 MMy2: GGC CAG GGG GAA AGA AAA A ... .., ....

... CC

... ... AT. ... ... ... .

5,854-872,864-888,848-872,1160-1184, 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 ... ..GS, 1369-1388, 1403-1421, 1369-1388, 1687-1706, 1651-1670, MMy28 AGG GCT GTT GGA AAT GTG G ... ... ... ... ..G ....

S, 2021-2039, 2055-2073, 2024-2042, 2329-2349, 2299-2318, I4My28bis: CCA CAT TTC CAG CAT CCC T ... ... ... .. G ....

... .. ... ... ..VS ... .

AS, 2039-2021, 2073-2055, 2042-2024, 2349-2329, 2318-2299 specific sequences of the vpr gene MMylB GAT AGA TGG AAC AAG CCC CAG S, 5590-5610, 5585-5605, 5554-5574, 6233- 6296, 6147-6170, FEUbaL.E DEZ. F? ECtIPLA - ', - "'. WrZE.... .... .. .. _S ,. ... .. 'I: ..., .. ..... .. ... ,.

W090 / 15066 206282 9 PCT / FR90 / 00393 8 MMy19: TCC ATT TCT TGC TCT CCT CTG T AS, 5870-5849, 5865-5844, 5834-5813, 6551-6531, 6454-6431, specific sequences of gene 1 MMy29 TAA AGC CAG GAA TGG ATG GCC CAA A.

... ... ... .... õ ..

... ...

S, 2620-2643, 2615-2638, 2584-2607, 2971-2994, 2887-3010 MMy29bis: TTG GGC CAT CCA TTC CTG GCT -.TA ..., ....>. . ...

... T.

AS, 2643-2620, 2638-2615, 2607-2584, 2994 = -2971, 3010-2887, MMy30: TGG ACT GTC AAT GAC ATA CAG AA ... ... ... ... ..T .. . ... ..

S, 3339-3361, 3334-3356, 3303-3325, 3690-3712, 3606-3628, MMy30bis: TTC TGT ATG TCA TTG ACA GTC CA ... ... ... ... ... .. T .....

AS, 3361-3339, 3356-3334, 3325-3303, 3712-3690 ,.

3628-3606, MMy31 CAT GGG TAC CAG CAC ACA AAG GS, 4186-4207, 4181-4202, 4150-4171, 4534-4555, 4450-4471, 2iy31bis: CCT TTG TGT GCT GGT ACC CAT G AS, 4207-4186, 4202-4181, 4171-4150, 4555 4534, 4471-4450, MMy32: TGG AAA GGT GAA GGG GCA GT .... ... ... ..A ..> ..

S, 4992-5011, 4987-5006, 4956-4975, 5340-5359, 5256-5275, MMy32bis ACT GCC CCT TCA CCT TTC CA ... ... ... ..T .õ .. ..

... ..,. ..C ..> ....>

AS, 5011-4992, 5006-4987, 4975-4956, 5359-5340, 5275-5256 FE Ã1j: mE Dc, P. EM P LA wFr- NfiE ~ i tiV0 90/3 5066 PïTI1FR90 / 00393 2062829 9 2 common sequences to the genomes of the 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-2 ROD and SIV-MAC viruses).

. specific sequences of the nef2 gene (encoding a negative factor of 27 kD) MMy12: AGA GAC TCT TGC GGG CGC GTG S, 9165-9185, 9139-9159, MR4Iy13: ATA TAC TTA GAA AAG GAA GAA GG S, 9542-9564, 9516-9538, MMyl3bis CCT TCT TCC TTT TCT AAG TAT AT AS, 9564-9542, 9538-9516, MMy14: AGC TGA GAC AGC AGG GAC TTT CCA AS, 9956-9933, 9893-9870, specific sequences of the vive2 gene (encoding for an infectivity factor of 23 kD) MMy20: TAT GGA GGA GGA AAA GAG ATG GAT AGT S, 5424-5450, 5340-5366, MMy21 TAG CAC TTA TTT CCC TTG CTT TS, 5754-5775, 5670-5691, MMy2lbis: AAA GCA AGG GAA ATA AGT GCT A AS, 5775 -5754, 5691-5670, MMy22 CCC TTG TTC ATC ATG CCA GTA T AS, 6082-6061, 5995-5974, specific sequences of the vpx gene (encoding a 12 kD protein) MMy23: ATG TCA GAT CCC AGG GAG AS, 5900-5918, 5813-5831, MMy24: CCT GGA GGG GGA GGA GGA GGA AS, 6228-6208, 6141-6121, 3) Sequences common to the genomes of the HIV-1 Bru, HIV-1 Mal, and HIV-1 Eli viruses (the series of figures spaced by a single line indicate the position of the FEUI ~~ DE E? ENqPLAt% "ëve-ro ~? -? NT WO 90/15066 PCT / FR90 / 00393 2062829 nucleotides on the genomes corresponding respectively to the viruses HIV-1 Bru, HIV-1 MAl and HIV -1 Eli).

specific sequences of the env gene (encoding the envelope proteins) MT3y5: CCA ATT CCC ATA CAT TAT TGT GCC CC S, 6905-6930,6903-6928,6860-6885 MMy5bis: GGG GCA CAA TAA TGT ATG GGA ATT GG AS, 6930-6905, 6928-6903, 6885-6860, MAiy6: AAT GGC AGT CTA GCA GAA GAA GA S, 7055-7077,7053-7075,7010-7032 PdMy7: ATC CTC AGG AGG GGA CCC AGA AAT TS, 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 MMy8bis: GGG TTC TTG GGA GCA GCA GGA AGC AC S, 7832-7857, 7821-7846, 7775-7800, 2SMy9: ATG GGT GGC AAG TGG TCA AAA AGT AG ... ... ... ..A ... ... ... ... ..

5,8844-8869, 8836-8861, 8787-8812, MMy9bis: CTA CTT TTT GAC CAC TTG CCA CCC AT AS, 8869-8844, 8861-8836, 8812-8787, MPIy78: TAT TAA CAA GAG ATG GTG GS, 7629 -7647, 7612-7630, 7572-7590, MMy89: CCA GCA AGA AAA GAA TGA AS, 8224-8242, 8213-8231, 8167-8185, MMy89bis: TTC ATT CTT TTC TTG CTG G AS, 8242-8224, 8231- 8213, 8185-8167, specific sequences of the nefl MASy10 gene: AAA AGA AAA GGG GGG ACT GGA S, 9116-9136, 9117-9137, 9062-9082, MMylObis: TCC AGT CCC CCC TTT TCT TTT AS, 9136-9116, 9137-9117, 9082-9062, i-Le WO 90/15066 2062829 PCë / pR90 / 00393 11 MMyll: AAA GTC CCC AGC GGA AAG TCC C AS, 9503-9483 , 9505-9484, 9449-9428, specific sequences of the alive gene 1 MMy15: GAT TAT GGA AAA CAG ATG GCA GGT GAT S, 5073-5099, 5068-5094, 5037-5063, MMy16: GCA GAC CAA CTA ATT CAT CTG TA S, 5383-5405, 5378-5400, 5347-5369, MMyl6bis: TAC AGA TGA ATT AGT TGG TCT GC AS, 5405-5383, 5400-5378, 5369-5347, MMy17 CTT AAG CTC CTC TAA AAG CTC TA AS, 5675-5653, 5670-5648, 5639-5617, specific sequences of the vpu gene MMy25: GTA AGT AGT ACA TGT AAT GCA ACC TS, 6081-6105, 6076-6100, 6045-6069, MMy26 AGC AGA AGA CAG TGG CCA TGA GAG S, 6240-6263, 6238-6261, 6207-6230, MMy27: ACT ACA GAT CAT CAA TAT CCC AA AS, 6343-6321, 6338-6316, 6307-6285, The invention also relates to the sequences (or primers) having a nucleotide structure complementary to those of the primers defined above.

It also relates to the nucleotide sequences exhibiting certain mutations with respect 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 affecting the hybridization properties of the sequences of the invention, can reach 40 ~.

In general, in the case of a sense (S) primer, a greater number of mutations will be tolerated on side 51 than on side 31 of the primer, the 3 'side having to hybridize perfectly. with a strand ~ REPL EUbLLE ~~ É.ZV: lEffT WO 90/15066 2062829 PCT / FR90 / 00393 12 determined of a nucleic acid sequence to allow amplification of this sequence.

In the case of an anti-sense primer (AS), the tolerance is: allowed on the 3 'side.

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

One of the characteristics of the oligonucleotide primers of the invention is to give a clear amplification band, generally devoid of nonspecific bands when the technical indications for use described in the present invention are implemented.

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 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 No. 88 / 307.102.9 of 08/01/1988.

A subject of the invention is in particular the use of the primers described above for the implementation of a method for the gene amplification of nucleic acid sequences of viruses of the HIV-1 and / or HIV-2, and / or F type. 'EUImLE t ~ E P.Eh7PLAe%. * WMl-rNT WO 90/15066 PCT / FR90 / 00393 13 SIV, this method being applicable to the in vitro diagnosis of the potential infection of an individual by a virus of the HIV-type 1 and / or HIV-2 or of an animal by at least one of the three viruses (HIV-1, HIV-2, SIV).

This in vitro diagnostic method of the invention is carried out from a batological sample (for example a biological fluid such as serum, lymphocytes from circulating blood) obtained from a patient under study, and comprises mainly the following steps - a step of extracting the nucleic acid to be detected belonging to the genome of the virus of the HIV-1 and / or HIV-2 and / or SIV type possibly present in the aforementioned biological sample, and, if applicable, 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 (the latter step being also referred to below as the step of reverse transcription of the 'A12N viral), - a cycle comprising the following steps 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 acid strands.

nucleic acid, obtained during the previous 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 the invention under the defined hybridization conditions below, formation from the primers of DNAs complementary to the strands on which they are hybridized in the presence of a polymerization agent (DNA polymerase) and of four different nucleoside triphosphate (dNTP), which leads to the formation of a FIRE: -L ~ DE â ~ EMPLAwEL7ENT WO 90/15066 c ~ PCT / FR90 / 00393 14 greater number of double-stranded nucleic acids to be detected than in the previous denaturation step , this cycle being repeated a determined number of times to obtain said nucleic acid sequence to be detected possibly present in the biological sample in a sufficient proportion to allow its detection, a step of detecting 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.

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

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, in particular inside blood cells, its RNA is copied into DNA by reverse transcriptase.

On the other hand, the genome of viruses of the HIV type in an extracellular medium, in particular in the blood, remains in the form of ARld.

The step of extraction according to the invention of the viral DNA contained in the cells of the biological sample recommended by the inventors - in addition to the conventional method using phenol chloroform - presents the following steps: suspension of the cell pellet in 0.5 ml of pyrolized water in a Potter large piston, crushing cells said by "back and forth", E'E UL LE. D E. R EM P ~~~ ew- M leE M r. . . . ',. . . . . . . '. ,. '.. .f ~. ... . . .

CA 02062829 2001-09-26 1 '90/15066 PCT / FR90 / 00'. 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 only cellular debris,. precipitation of the DNA 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 70 'ethanol.

It should be noted that this method allows the joint precipitation of DNA and RNA, which allows the detection of the genomic message of viruses of the HIV or SIV types by using the method known as "direct DNA PCR" or by that known as "PCR. -RNA ".

The step of extracting the viral RNA 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-stranded into double-stranded DNA is necessary to be carried out when 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, 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.

A more particular subject of the invention is, inter alia, an in vitro diagnostic method as defined above, in which the step of reverse transcription of the viral RNA is carried out as follows * Trademark Fc.U ~ _LE DE REPLaC -.EME ~ M WO 90/15066 PCr / FR90 / 00393 2062829 16 - 10 g of extracted RNA resuspended in water is placed in the presence of the pair of primers at a concentration of 40 MM each, in a final volume of 40 Ml. The whole is denatured at 100 ° C. for 10 minutes then immersed in ice-cold water, - 10 l of the following mixture are added: 5 l of the "10 X buffer" described below + 1 unit of reverse -transcriptase (from AMV (Avian Myeloblastosis Virus) or MuMLV (Moloney Leukemia virus)) + 1 unit of Taq-polymerase + 1 41 of the mixture of 4 dNTPs at 25 mM each + QSP water 10 Ml.

The final volume is therefore 50 Ml.

This reaction is carried out in two stages:

- a) 1st step: manufacture of the cDNA by the action of reverse transcriptase at 42 ° C for 13 minutes, - b) 2nd step: classic 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 is started.

A more particular subject of the invention is an in vitro diagnostic method as described above, in which the denaturation step is carried out in the presence of the pair (s) of primers (or primers) of the invention.

Indeed, as it was 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 :

- hybridization: the primers (1 1 of a 40 molar solution (40 4M) of each primer) are placed in the presence of the DNA-template (100 to 300 ng) for the first stage of denaturation-reassociation; heating for 10 minutes at 100 ° C then immersing the tubes containing this FEUbLLE DE RoErb4PL ~ IC EENI m- T7 WO 90/15066 PL'I '/ F'R90 / 00393 fa ~ â ~ / dOF + ~ 17 mixture of 'DNA-matrix and primers in ice-containing water in order to increase the rate of DNA-matrix reassociation / primers.

The primers should be used at a final concentration in the subsequent amplification step of 0.8 µM each.

- Amplification: the 4 A dNTPs are added to the above medium, each being used at 0.5 pmolar in final solution (50 l), and one unit of Taq-polymerase for a reaction medium of 50 l; this step is carried out in an amplification buffer of the present invention, generally designated under the name of 1110 X buffer ", the composition of which (when it is diluted to 1/10) is as follows: Tris-HC1, pH 8, 9: 50 mM;

(NH4) Z SO4: 15 mM; MgCl2: 5 mM; P-mercapto-ethanol:

mM; gelatin: 0.25 mg / ml.

5 l of this buffer and water qs 50 41 are added 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 will be followed by a single cycle at 78 C for 15 minutes.

The precision of the temperatures indicated to within 0.36C, 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 optimum 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 41, and that these FcU conditions:; ~ LE DE wO 90/15066 s ~ Q62829 PCT / FR90 / 00393 ~ + 18 can be modified depending on the final volume of the reaction medium.

The use of several pairs of different primers (or cocktails of pairs) of the invention allows either the cross detection of several types of virus of the HIV and / or SIV type, or 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, MMy2-Mhiy4, MMy1-MPRy3, MMylB-MMy19, MMy4bis-MMy28bis, MMy28-MMy29bis, MMy29-MMy30bis, MMy3l-MMy32bis, in particular for the in vitro diagnosis of the infection of an individual by HIV-1 and / or HIV-2 - MMy5-MMy8, MMy6-MMyB, MMy7-MMy8, MMy5 -MMy7bis, MMy6MMy7bis, MMy9-MMyll, MMylO-Myll, MMy9-MMylObis, MMy26MMy5bis, MMy8bis-MMy9bis, MMyBbis-M4iy89, MMy89bisMMy9bis, MMyl5-MMyl7, MMy15-MMyl6bis, MMy16-MtRyl7 ,.

MMy25-MMy27, MMy26-MMy27, in particular for the in vitro diagnosis of the infection of an individual by HIV-1, - MMy20-MMy22, MMy20-MMy2lbis, MMy21-MMy22, MMy23MMy24, MMy12-MMyl4, MMy12-MMyl3bis, for the in vitro diagnosis of the infection of an individual with HIV-2.

The polymerization agent used in the step of extending the cycle is a thermostable DNA polymerase, in particular Taq polymerase, amplifiose from the firm of Appligene or any thermostable DNA polymerase which can be marketed.

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

The in vitro diagnostic method of the invention also makes it possible, depending on the pairs of nucleotide primers used, to selectively detect the SHEETS OF ;. = WO 90/15066 PCr / FR90 / 00393 19 genes for viruses of the HIV and / or SIV type present in the biological sample.

The pairs of primers which can be used, by way of examples, for the aforementioned gene-by-gene diagraostic method of the invention are as follows - MMyl-MMy4, MMy2-MMy4, MMyl-34My2l, MFYy4bis-AMy28bis for the gag gene, - MMy18-MMy19 for the vpr gene, - Mbiy5-MMy8, 2RMy6-riMyB, MMy7-MMy8, 24Piy5-MDiy7bis, MMy6MMy7bis, MMy26-MNIy5bis, MMy8bis-MMy9bis, N + iyBybis-MNty -89, N + iyB9bis-MNty -89, env gene, - kIIMy9-MMyll, MMy9-MMylObis, MMy10-T4My11 for the nefl gene, -> Oviyl5-hMy17, Mr3y15-PR + Syl6bis, M4iy16-MMy17, for the vivel gene, - NR + ly20-2RMy22, MMy2Q-MMy2lbis, MMy21-MMy22 for vive 2, - MMy23-AMy24 for vpx, - MMy12-MMy14, MMy12-I4riyl3bis, AMy13 -MAiy14 for nef2, - MMy25-NRMy27, MMY2 6-MMy27 for the vpu gene, - MMy28-I4Z + Iy29bis, MMy29-MMy3Obis, MMy3O-MMy3lbis, MMy31-MMy32bis for the pol gene.

However, the combinations between “S” and “AS” priiners described above are not limiting and can be varied according to the desire of the user.

The sizes of the nucleotide fragments synthesized using the pairs of primers mentioned above by way of example, are indicated in the following Tables I to XI (the figures indicated in the tables 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).

Fc ~ âIL ~~ 4VO 90/15066 PCT / FIt90 / 00393 Table I ------------------ .. -----------..- ---------., ---..------ gag. gag ------------------- ------------ ---- -------------- -: Mpiy1-MMy3: riNlyl-MMy4: Mriiy2 -MAiy4: MMy4bis-MMy2 8bis:

----------------- --------------------------------- --- HIV1-BRU: 265. 750. 532 = 671 ------------------------------------ ----------- -------- HIV1-MAL: 282. 785,556. 671 ------------------------------------------------- ---- HIV1-ELI: 265. 750 = 538. 674 ----------------------------------------------, - ----- HIV2-ROD: 354. 845. 544. 663 SIV: 343. 844 = 544. 668 ------------------------------------------------- ---- Table II -------------------------------------------- --------- approx. env ------------------------------------------------- ------: MMy5-MMy7bis: Agiy5-MMy8: MMy6-MMy7bis: MMy6-MMy8:

-------------------------------------------------- ---- HIV1-BRU: 480. 953. 330. 803 ---------------------------..---------- - ..------- -., - HIV1-M ~ L: 471 944. 321. 794 ----- ----------- -----------------..-------------- --- HIV1-ELI: 471. 941, 321 791 ---------------------------------------------- ------- HIV2-ROU:

-------------------------------------------------- ---IF V ---------------------------------------------- -------- FE ~ rmLi D EL RE? 'T7PL.AE WO 90/15066 2062829 PCT / RR90 / 00393 21 Table III ------------------ --- --------------, ------------------ ,.

env env ------------------------------------, - -a - - - - -: 24riy7-Agiy8: MMy2 6-MMy5bis: PqMy8bis-.MMy9bis ---------------------------------- - ----------------- HIV1-BRU: 498. 691. 1038, --------------------.. ---------------. ---- ..---- -_------ HIV1-MAL: 498. 691. 1041.

-------------------------------------------- HIV1-ELI: 495 679 1038>

-------------------- _ ---- _ ----- ..- -----.. --- _ - __-- _-HIV2-ROD: -, -, -, ------------------------------..------ --------------- IF V . -, -. - ~ --------------------..-------------------------- ----- FEUI; L: GE RE ~~ P LAP.7 ~~ WO 90/15056 2062829 PCT / FR90 / 00393 22 Table IV ------------------------- ----------------------------- approx. env ------------------------------------------------- ------: MMy8bi.s-I4My89: MMyE39bis-HMy9bia ---------------------------------- -._------------------ HIV1-BRU: 411. 646 ------------------------------------.------------ -..--- HIV1-MAL: 411. 649.

-------------------------------------------------- --- HIV1-ELI: 411. 646 ------------------------------------------------- ---- HIV2-ROD:

-------------------------------------------------- ---IF V ---------------------------------------------- ------- Table V ----------------------------------------- ------------ nefl nef3 ------------------------------------ -----------------: MMy9-MMylObis: AiMy9-Y4my11: 24riy10-MMy11 --------------------- -------------------------------- HIV1-BRU: 293, 660. 388.

-------------------------------------------------- --- HIV1-MAL: 302. 660. 388 ------------------------------------------------- --- HIVl-ELI: 296 663 388 ---------------------------------------- ------------ HIV2-ROD:

-------------------------------------------------- ---IF V , - . -. -.

-------------------------------------------------- --- REM PLACCE SHEET.

Pe. ~ Ii WO 90/15066 PCI '/ k Ft90 / 00393 2062829 23 Table VI --------------------------- - --- - ------------------ nef2 nef2.

---------------------------------- ---------------- ---- M4iy12-MMyl3bis: MMy12-lMMy14: MMy13 -MMy14 ----------------------------------- -------------------- HIV1-BRU: -, -. -, ---------------------------- ----...------- ----- ------ HIV1-MAL: -. -, -, ---------------------------------------------- ---- - .. HIV1-ELI: -, -, ----------------------- a ----------- ------------------ HIV2-ROD: 400, 792. 415 SIV: 400. 755. 378 -------------------------------------------------- --- Table VII --------------------------------------------- -------- Vividl Vivid3 ---------------------------------------- --------------: MMy15-MMyl6bis: MMy15-MMy17: MY.y16-MMy17.

-------------------------------------------------- ---- HIV1-BRtJ: 333. 603. 293 ------------------------------------------------- ---- HIV1-MAL: 333. 603 293 ------------------------------------------------ ----- HIV1-RLI: 333. 603. 293 ------------------------------------------------- ---- HIV2-ROD: -. -, -, ---------------------------------------------- ------- IF V . -> -. -.

-------------------------------------------------- --- ~ EU 1-LE DE REPL.A ~; Lvà-Fe-3M w 90/15066 F'C "I '/ FR90 / 00393 24 Table VIII ------------ ----------------------------------------- vpr. vive2 __________________________________.___- -__ ----__-_-_-__: MrYy18-I + My19: MMy20-MFiy2lbie: A4Sy20-MMy22 ------------------------- --------- .o -_ - _ - _ - _-_ a __-___ HIV1-BRU: 281. -, -, _____________________________________________________o HIV1-MAL: 281. -. -, ------------------------------------------------ ----- HIV1-ELI: 281 ---------------------------------------- ------------- HIV2-ROD: 319. 352 659 ________________________.. ------- ____-_-__ - _-..__-___--:

SIV: 308. 352. 656 ------------------------------------------------- ---- Table IX -------------------------------------------- --------- vivid2, vpx -------------------------------------- ---------------- MMy21-P ~ iy22 MMy23-MMy24 __ ------- _-_--- ..-__-_ - ..-- --------------------..__--- HIV1-BRU: -, -.

-------------------------------------------------- --- HIVl-MAL: ------------------------------------------- ---------- HIV1-ELI: - _, --------------------------------- -------------------- HIV2-ROD: 329 329 ------------------------ ----------------------------- SIV: 326. 329 , ------------------------------------------------- ---- ~ E Ub ~ .L ~ ~ E REPL, A ~ EME WO 90/15066 PCT / PR90 / 00393 Table X --------------------- ------------- _------------------ vpu, pol --- ------------ ------------------._-------------------: MMy25-MAiy27: MMy26-MMy27: MMy28- MMy29bis -----------------.._ --- _-.. --------- .------------ --_----- HIV1-BRU: 263. 104. 623 ----------------------------------. M-- --..------- ---- HIV1-MAL: 263. 101. 584 -------------------------------------------------- --- HIV1-ELI: 263. 101. 584 ------------------- ------------ ..-------- ---..--- ..---- HIV2-ROD: 666 -.. ------- .. --------------------- ..---- -.. ------ ..-------- SIV 712 ----------------------------- ----------------------- Table XI ------------------------- ---------------------------- Pol. P 1 -----------------------------------------: MMy29-14riy3obi.s: MMy30-M2PRy31bis: 1 ~ -My31-MNiy32bis ---------------------------------------- -------------- HIV1-BRU: 742. 869. 826 ------------------------------------------------- ---- HIV1-MA.L: 742: 869. 826 ------------------------------------------------- ---- HIV1-ELI: 742. 869. 826 ------------------------------------------------- ---- HIV2-ROD: 742. 866. 826 ------------------------------------------------- ---- SIV: 742. 866. 826 -------------------------------------------------- --- ~~~ l; s ~ i Gr RE ~~ PLACEl ~ .iEi ~ 1u'O 90 / d 5066 PCf / FR90 / 00393 ---, 26 It should be noted that thanks to their arrangement on the genome, the primers used for amplification can be combined in such a way that they can be used as a probe, either after labeling with 32 p by kination, or for use in the cold probe technique to verify the specificity of the amplification band observed during a "Southern blot" analysis.

In addition to the conventional combination of primers so that a third oligonucleotide can serve as a specific internal probe, it should be noted the particular case of the vivelJvpr and vive2Jvpx genes due to the overlap of these genes, which allows cross-detection.

In addition, during an analysis by sequencing of the amplified DNA, these oligonucleotides can be used as a specific primer for the DNA polymerase allowing a double sequencing in each direction, therefore a double reading of the sequences, thus removing any possible ambiguities. 'interpretation.

A subject of the invention is also the primers, as defined above, labeled, in particular radioactively or enzymatically, as well as their use as nucleotide probes, in particular in the context of the in vitro diagnostic method as described. above.

A subject of the invention is also oligonucleotides as described above and comprising sugars in α conformation.

Such oligonucleotides have the characteristic of reversing the direction of the double helix formed with the template (strand of the virus genome), this double helix thus passing from the nS "state to the" AS "state.

The invention also relates to the ol.igonucleotides described above, some nucleotides of which are methylated and / or contain one or more atoms of FGU ~ LLs DE R1EMPLACCE P. ~ "41 '90115t} fi6 PCT / ~~ MM ~ 27 sulfur in particular on adenines.

Such oligonucleotides have the characteristic of increasing the stability of the double helix, and consequently of better hybridizing with the strand of Al3N to be amplified.

The invention also relates to oligonucleotides as described above and in the so-called “modified bases” form comprising nucleotides onto 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 appeared in "La Vie des Sciences", reports, general series, volume 4, n'l, p. 17-37.

Such oligonucleotides have the characteristic of being easily detectable, in particular by fluorescence.

The oligonucleotides 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 SIV type, this method comprising the main characteristics of that described above.

A subject of the invention is also 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 be detected, and a primer hybridizing with the complementary strand of this last under the conditions defined above, EEUb; ~~ DE P.EN4PLA :: vb-P.IENT Wo 90/15066 PC '/ F'R90 / 00393 2062829 28 - reagents suitable for carrying out the cycle of amplification operations, in particular DNA polymerase, and four different triphosphate nucleotides, and the reaction medium called 1110 X buffer "described above.

one (or more) probe which can be labeled, in particular by radioactivity or by the cold probe technique, capable of hybridizing specifically with the amplified nucleic acid sequence (s) to be detected.

The invention also 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.

By way of illustration, this protein synthesis process comprises the amplification of nucleotide sequences of the genomes of viruses of type IiZV or SIV (coding for a determined protein and having undergone, where appropriate, certain modifications of their nucleotides) by bringing into contact said sequences with at least one pair of primers according to the invention under the conditions described above, followed by the translation of these sequences thus amplified into proteins; this last step is carried out in particular by transformation of suitable host cells using vectors containing said amplified sequences, and recovery of the proteins produced in these host cells.

The invention also relates to the polypeptides resulting from the translation of the nucleotide sequences (or primers) of the invention.

A subject of the invention is also the use of antisense oligonucleotide primers as antiviral agents in general, in particular in the EEUÃLLE DE F? EMPLA *% "o * ca-'R ~ ENT WO 90/15066 PC ' i '/ FR90 / 00393 2062829 29 fight against AIDS, as well as pharmaceutical compositions containing these antisense primers in association with a pharmaceutically acceptable vehicle.

The invention also relates to 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 invention relates to 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 products of translation of the nucleotide sequences according to the invention, or one or more translation products of the nucleotide sequences amplified from the primers defined according to the invention) and their use for the implementation of in vitro diagnostic methods of the infection of an individual by a virus of the HIV type -1 and / or HIV-2, or of an animal by at least one of the three viruses (HIV-1, HIV-2, SIV) according to the methods known to those skilled in the art.

By way of illustration, such an in vitro diagnostic method according to the invention comprises bringing a biological sample (in particular serum) taken from a patient under study into contact with antibodies according to the invention, and the detection using any suitable method (in particular using labeled antiimmunoglobulins) of the immunological complexes formed between the antigens of the viruses of the HIV or SIV type FIRE: LLi DE ~ EMPLA ~ EMENf WO 90/25066 PCi / FR90 / 00393 2062829 possibly present in the biological sample and said antibodies.

A subject of the invention is also in vitro diagnostic kits comprising antibodies according to the invention and, where appropriate, reagents suitable for demonstrating the immunological reaction formed between said antibodies and the antigens of the HIV or SIV viruses. .

The invention also relates to a process for preparing the polypeptides mentioned above, in particular those corresponding according to the universal genetic code to the nucleotidic sequences (or primers) described above, this process being characterized in that, preferably starting from the C-terminal amino acid, the successive aminoacyls are condensed two by two in the required order, or aminoacyls and fragments formed beforehand and already containing several aminoacyl residues in the appropriate order, or again several fragments thus prepared beforehand, it being understood that care will have been taken to protect beforehand all the reactive functions carried by these aminoacyls or fragment with the exception of the amine functions of one and the carboxyl of the other or vice versa, which should normally be involved in the formation of peptide bonds, in particular after activation of the carboxyl function, according to the methods known in the synthesis of peptides and so on, step by step, up to the N-terminal amino acid.

For example, recourse will be had to the technique of peptide synthesis in homogeneous solution described by Houbenweyl in "ASeuthode der organischen Chemie" (Method of Organic Chemistry) edited by E.

Wunsch, vol. 15-I and II, THIEME, STUTTGART, 1974, or to that of solid phase peptide synthesis described by RD

~ EUILL. ~ ~~ REPLACEMENT eilo 90/15066 PCX '/ Fdt90 / 00393 2062829 31 Merrifield in "Solid Phase Peptide Synthesis" (J. AM.

CHEM. SOC., 45, 2149-2154).

The invention also relates to a process for preparing the nucleotide sequences (or primers) described above, this process comprising the following steps:

- incubation of the genomic DNA, isolated from one of the viruses of the HIV or SIV type mentioned above, with DNAse I, then addition of EDTA and purification by extraction with a phenol / chloroform / isoamyl alcohol mixture ( 25/24/1) then with ether, treatment of the DNA thus extracted with Eco Ri methylase in the presence of DTT, and purification by extraction as described above, - 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 from E. coli, then purification according to the method described above, the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the desired nucleic acid at the same time. using an appropriate probe.

A particularly advantageous process for preparing the nucleotide sequences of the invention comprises the following steps - DNA synthesis using the automated method of P-cyanethyl phosphoramidite described in Bioorganic Chemistry 4; 274-325 (1986), - the cloning of the nucleic acids thus obtained in a suitable vector and the recovery of the nucleic acid by hybridization with a suitable probe.

Another process for preparing the nucleotide sequences of the invention comprises the following steps ~ EWmLE DE RENI PLACZEME ~ MW 90/15066 P (? / F'R90 / 00393 2062829 32 - the assembly of chemically synthesized oligonucleotides, provided with their ends of different restriction sites, the sequences of which are compatible with the amino acid sequence of the natural polypeptide according to the principle described in Proc.

Natl.

Acad. 8c2, USA, 80; 7461-7465, (1983), - the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the desired nucleic acid by hybridization with an appropriate probe.

F ECU ~~ .l ~~ DE FiENiP1 ~ ACVN3E1 ~ i