Oligonucleotide sequences for amplification of type HIV-2 and siv retroviruses genomes and their application to in-vitro diagnostic of infections caused by these viruses.

The present invention relates to oligonucleotide sequences which can be used in particular as oligonucleotide primers for the implementation of techniques for amplifying nucleic acid sequences of human immunodeficiency retroviruses of HIV-2 type, or of monkey immunodeficiency retroviruses. SIV type.

The invention relates in particular to the application of these sequences to diagnostic methods <u style="single">in vitro</u> in humans, the infection of an individual by a retrovirus of the HIV-2 type.

The isolation and characterization of retroviruses grouped under the designation HIV-2 were described in European patent application n^o 87 / 400.151.4. These retroviruses have been isolated from several African 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 respect to these lymphocytes when they multiply therein, thereby causing either 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" (possibly immunodeficiency virus of the monkey) possibly followed by an abbreviation designating the species of monkey from which they come, for example "mac" for the macaque "or" AGM "for the green African monkey (abbreviation of" African Green Monkey ").

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

Studies have shown that the SIV-1 retrovirus contains certain proteins which have an immunological relationship with proteins or glycoproteins capable of being obtained under analogous conditions from HIV-2.

The pursuit of the study of HIV-2 retroviruses has also led to the obtaining 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 n^o I-532, under the reference name LAV-2 ROD.

Diagnostic methods <u style="single">in vitro</u> infections with viruses of the HIV-2 type currently existing, most often call for the detection of anti-HIV-2 antibodies possibly present 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-2, under conditions allowing the production of a possible immunological reaction of these extracts or antigens with these antibodies.

Such diagnostic methods are not very sensitive and are likely to be false negative, in particular in the case of a recent infection of an individual by the HIV-2 virus.

Genomic amplification techniques are a considerable adjunct to the development of diagnostic methods <u style="single">in vitro</u> particularly susceptible to viral diseases. Among these amplification techniques, mention may be made of the PCR (Polymerase Chain Reaction) technique as described in European patent applications n^o 86 / 302.298.4 dated 03/27/1986 and n^o 87 / 300.203.4 of 09/01/1987, or the so-called "Qβreplicase" technique described in Biotechnology, vol. 6, page 1197 (October 1988), and that using a RNA polymerase (T7 RNA polymerase) described in international patent application n^o WO89 / 01050. They make it possible to improve the detection sensitivity of nucleic acids of viruses, but however require the use of specific synthetic primers.

For the detection of viruses of the HIV-2 type, 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 HIV-2 isolate may fail to amplify certain viral variants of the HIV-2 type. On the other hand, even if a primer is chosen in a conserved region of the genome of an HIV-2 virus to another, its "good functioning" is not guaranteed and can give rise to poor yields of amplification.

The present invention precisely provides oligonucleotide sequences (or primers) allowing the amplification for diagnostic purposes of the genome of all viruses of the HIV-2 type, with good yields.

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

The present invention relates to oligonucleotide primers, of approximately 15 to 25 nucleotides, which can be used for the genomic amplification of viruses of the HIV-2 or SIV type.

A primer according to the invention is characterized in that its nucleotide sequence is: - either chosen from those contained in one of the nucleic sequences delimited by the nucleotides located at positions 40 and 61, 9537 and 9558, 240 and 259, 546 and 569, 906 and 927, 612 and 633, 1857 and 1876, 2078 and 2101, 6275 and 6299, 6855 and 6878, 7548 and 7573, 7782 and 7805, 8412 and 8434, 61 and 82, 9558 and 9579, cDNA derived from the genome of the HIV-2 ROD virus, or which are complementary one of the above-mentioned nucleic acid sequences, - either chosen from those which are contained in one of the nucleic sequences delimited by the nucleotides located at positions 40 and 61, 9511 and 9532, 240 and 259, 256 and 275, 551 and 574, 911 and 932, 617 and 638, 1868 and 1887, 2035 and 2058, 6227 and 6251, 7550 and 7564, 7776 and 7799, 8406 and 8428, 61 and 82, 9532 and 9553, cDNA derived from the genome of the virus SIVmac 142, or which are complementary to l 'one of the above-mentioned nucleic acid sequences, - Either (in particular for the longest primers) contains one of the abovementioned nucleic sequences derived from HIV-2 ROD or from SIVmac 142, or contains a nucleic sequence complementary to one of these latter sequences, it being understood that the nucleotides any additional which "extend" over the nucleic sequence of the genus in question, preferably on the 5 ′ end side, preferably coincide with those which are placed below the corresponding 5 ′ end within the complete sequence of HIV-2 ROD or SIVmac 142, - it being also understood that the strands of the cDNAs which are taken into consideration are those which are found to be complementary to the RNAs of the HIV-2 ROD and SIVmac 142 viruses, - 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, nevertheless capable of hybridizing with this nucleic sequence derived from the HIV-2ROD and / or SIVmac 142 viruses mentioned above in a solution composed of 10 mM Tris, 20 mM KCl, 2 mM MgCl₂, and 0.01% gelatin, during 1 minute at a temperature greater than or equal to 50 ° C.

The numbering of the nucleotides mentioned above corresponds to that used in FIG. 2 of the article by GUYADER et al, Nature, vol. 326, p. 662-669 (1987) as regards the HIV-2 ROD cDNA, and that used in FIG. 1 of the article by CHAKRABARTI L. et al, Nature, vol. 328, p. 543-547 (1987) for the cDNA of SIVmac 142.

The invention relates more particularly to the oligonucleotide primers characterized by the following nucleotide sequences (represented in the direction 5 ′ → 3 ′:<img file="EP0404625A2_D0001.tif" />

The above-mentioned primers are identical or complementary to the following nucleic acid sequences, derived from the cDNA of the HIV-2 ROD virus or that of the SIVmac 142 virus: - the primer LTR1 is identical on the one hand, to the nucleic sequence comprising the nucleotides located at positions 40 to 61, as well as to that comprising the nucleotides located at positions 9537 to 9558 of the HIV-2 ROD cDNA , and, on the other hand, to the nucleic sequence comprising the nucleotides located at positions 40 to 61, as well as to that comprising the nucleotides located at positions 9511 to 9532 of the cDNA of SIVmac 142, the primer LTR2 is complementary on the one hand, nucleic sequences comprising the nucleotides located at positions 240 to 259 of the HIV-2 ROD and SIVmac 142 cDNAs, and, on the other hand, the nucleic sequence comprising the nucleotides located at positions 256 to 275 of the SIVmac 142 cDNA , the primer GAG1 is identical to the nucleic sequence comprising nucleotides 546 to 569 of the HIV-2 ROD cDNA, and to the nucleic sequence comprising the nucleotides located at positions 551 to 574 of the SIVmac 142 cDNA, the primer GAG5 is complementary to the nucleic sequences comprising the nucleotides located respectively at positions 906 to 927 and 911 to 932 of the cDNAs of HIV-2 ROD and SIVmac 142, the primer GAG2 is identical to the nucleic sequences comprising the nucleotides located respectively at positions 612 to 633 and 617 to 638 of the cDNAs of HIV2 ROD and SIVmac 142, the primer GAG2B is complementary to the above-mentioned nucleic acid sequences identical to GAG2, the primer POL1 is identical to the nucleic sequences comprising the nucleotides located respectively at positions 1857 to 1876, and 1868 to 1887 of the cDNAs of HIV-2 ROD and SIVmac 142, the primer P1 is identical to the nucleic sequences comprising the nucleotides located respectively at positions 6275 to 6299, and 6227 to 6251 of the cDNAs of HIV-2 ROD and SIVmac 142, the primer P2 is complementary to the nucleic sequence comprising the nucleotides located at positions 6855 to 6878 of the HIV-2 ROD cDNA, the primer P2B is complementary to the nucleic sequence identical to the above-mentioned P2, the primer P4 is complementary to the nucleic sequence comprising the nucleotides located at positions 7548 to 7573 of the HIV-2 ROD cDNA, and partially complementary to the nucleic sequence comprising the nucleotides located at positions 7550 to 7564 of the cDNA of SIVmac 142, the primer P6 is identical to the nucleic sequences comprising the nucleotides located respectively at positions 7782 to 7805, and 7776 to 7799 of the cDNAs of HIV-2 ROD and SIVmac 142, the primer P7 is identical to the nucleic sequences comprising the nucleotides located respectively at positions 8412 to 8434 and 8406 to 8428 of the HIV-2 ROD and SIVmac 142 cDNAs, the primer P7B is complementary to the nucleic sequences identical to the primer P7 mentioned above, the primer P8 is complementary on the one hand, to the nucleic sequence comprising the nucleotides located at positions 61 to 82, as well as to that comprising those located at positions 9558 to 9579 of the HIV-2 ROD cDNA, and on the other hand, to the nucleic sequence comprising the nucleotides located at positions 61 to 82, as well as to that comprising the nucleotides located at positions 9532 to 9553 of the cDNA of SIVmac 142.

A subject of the invention is also the primers having a nucleotide structure complementary to those of the primers LTR1, LTR2, GAG1, GAG5, POL1, POL2, P1, P4, and P8 defined above.

It also relates to primers having certain mutations compared 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, is generally between 0% and 10%, and preferably does not exceed 20% .

In general, 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.

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 genomic amplification method by synthesis of multiple copies of RNA as described in European patent application n^o 88 / 307.102.9 dated 08/01/1988.

The invention more particularly relates to the use of the primers described above for the implementation of a diagnostic method <u style="single">in vitro</u> infection of an individual with an HIV-2 virus.

This diagnostic method <u style="single">in vitro</u> of the invention is carried out from 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 extracting the nucleic acid to be detected belonging to the genome of the HIV-2 type virus possibly present in the abovementioned 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 in order to obtain a double-stranded nucleic acid, - a cycle comprising the following stages: . denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid, . hybridization of each of the nucleic acid strands, obtained during the preceding denaturation step, with at least one primer according to the invention, by bringing the above-mentioned strands into contact with at least one pair of primers according to invention under the hybridization conditions defined above, . formation, from the primers, of DNAs complementary to the strands on which they are hybridized (elongation step) in the presence of a polymerization agent and of four different nucleoside triphosphate, which leads to the formation of a larger number 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, a step of detecting the possible presence of the nucleic acid belonging to the genome of the HIV-2 type virus in the biological sample.

The diagnostic method <u style="single">in vitro</u> of the invention can be carried out either from RNA or from viral DNA.

Indeed, the genomes of HIV-2 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 the HIV-2 type viruses in an extracellular medium, in particular in the blood, remains in the form of RNA.

The step of extracting the viral DNA contained in the cells of the biological sample is more particularly detailed in the article by LAURE F. et al, published in Lancet, p. 538-540 (1988).

By way of illustration, the lymphocytes are separated from the other blood constituents by centrifugation in a Ficoll gradient. The lymphocytes thus obtained are then treated with a lysis buffer consisting of 10 mM pH 8 tris, 10 mM EDTA, 10 mM NaCl, 0.5% SDS (sodium dodecylsulfate) and 100 μg / ml of proteinase K for 2 hours at 60 ° C. the DNA is then extracted with phenol and then precipitated with ethanol.

The extraction can also be carried out in an identical manner to that previously described, on the concentrated serum. In this case, RNA is obtained and an additional step of transformation of single-strand RNA into double-stranded DNA is necessary to perform when the diagnosis<u style="single">in vitro</u> of the invention is carried out from biological samples containing viruses of the HIV-2 type 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 the reverse transcriptase enzyme, under the conditions indicated by the supplier (Amersham for example).

In a preferred embodiment of the diagnostic method of the invention, the cycle denaturation step is carried out for 1 minute at 94 ° C.

The hybridization stage of the diagnostic method cycle <u style="single">in vitro</u> of the invention is advantageously carried out by bringing the nucleic acid strands obtained during the denaturation step of the cycle into contact with at least one pair of primers of the invention, these primers being chosen so that the 'one of these two primers hybridizes with a nucleic sequence located on one of the two strands while the other hybridizes with a nucleic sequence located on a complementary strand of the latter, said nucleic acid sequences (with which said primers are liable to hybridize) being separated, when considering the two complementary strands mentioned above grouped together in a double-stranded nucleic acid, by a number of base pairs between 50 and 10,000 , preferably between 100 and 2000.

The use of several pairs of different primers of the invention allows the amplification and the detection of nucleic acids different from the HIV-2 genome.

By way of example of pairs of preferred primers which can be used in the context of the present invention, mention may be made of primers LTR1 and GAG2. Mention may also be made of the pairs P1 and P2, P2 and P7, P7 and P8, P8 and LTR2. Advantageously, the pairs of primers used are chosen so that the DNA fragments synthesized cover the regions P1 to P2, LTR1 to Pol2, P2 to P7, P7 to P8, P8 to LTR2.

The polymerization agent used in the elongation stage of the cycle is a DNA polymerase, in particular Taq polymerase or even any polymerase suitable for implementing a diagnostic method <u style="single">in vitro</u> according to the invention according to the principle of the "QβReplicase" technique or that described in the above-mentioned international patent application.

Generally speaking, the diagnostic method cycle <u style="single">in vitro</u> of the invention is repeated between 10 and 60 times, and preferably 40 times.

Advantageously, the cycle elongation step of the above-mentioned method of the invention is carried out for 1 minute at 72 ° C.

For example, for 1 μg of the retroviral DNA to be detected, 10 pmol of each primer, 10 nanomoles of each nucleotide triphosphate (dTNP), 1 U of Taq polymerase are used in a final volume of 100 μl of the buffer: Tris 10 mM pH 8.3 (measured at 23 ° C) KCl 20 mM MgCl₂ 2 mM 0.01% gelatin and subjected 40 times to the following thermal cycle: 1 min at 94 ° C (denaturation) 1 min at around 55-60 ° C (hybridization) 1 min at 72 ° C (elongation)

In a preferred embodiment of the diagnostic method <u style="single">in vitro</u> of the present invention, the step of detecting the possible presence of the nucleic acid of the HIV-2 type virus in the biological sample is carried out using one (or more) labeled nucleotide probe capable of s hybridize with the amplified nucleic acid sequence (s) and in that any hybridization complexes then formed are detected between the probe (s) and the amplified nucleotide sequence (s) to be detected.

A 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 diagnostic method. <u style="single">in vitro</u> as described above.

The primers of the invention can also be used for the implementation of a diagnostic method <u style="single">in vitro</u> infection of monkeys (macaque, mangabeys monkey or green monkey) by the virus of the SIV type, this method incorporating the main characteristics of that described above.

The subject of the invention is also diagnostic kits for implementing diagnostic methods <u style="single">in vitro</u> mentioned above. 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, - reagents suitable for the implementation of the amplification operation cycle, in particular DNA polymerase, and four different triphosphate nucleotides. - one (or more) labeled probe capable of hybridizing with the amplified nucleic acid sequence (s) to be detected.

The invention also relates to a method for synthesizing the primers described above.

The invention also relates to a process for the production of one (or more) peptide (or polypeptide) comprising: a step of amplifying the nucleic sequence coding for this peptide (and advantageously containing a promoter for the translation of this sequence) using a pair of primers according to the invention, the introduction of said nucleic sequence thus amplified into an appropriate vector, - transformation of appropriate host cells using the above-mentioned vector, - culturing the host cells thus transformed and recovering the peptide produced by them.

The invention also relates to the polypeptides corresponding, according to the universal genetic code, to the nucleotide sequences (or primers) described above.

The invention also relates to the use of the above-mentioned peptides as immunogenic agents, in particular in combination with a pharmaceutically acceptable vehicle in a pharmaceutical composition.

The invention also relates to a process for the preparation of the above-mentioned polypeptides, in particular those corresponding according to the universal genetic code to the nucleotide sequences (or primers) described above, this process being characterized in that, preferably starting from the C-terminal amino acid, the successive aminoacyles are successively condensed two by two in the required order, or aminoacyls and fragments previously formed and already containing several aminoacyl residues in the appropriate order, or several fragments previously thus prepared, it being understood that care has been taken beforehand to protect all the reactive functions carried by these aminoacyles or fragment with the exception of the amine functions of one and the carboxyl of the other or vice versa, which must normally intervene 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, we will use the peptide synthesis technique in homogeneous solution described by Houbenweyl in "Meuthode der Organischen Chemie" (Method of Organic Chemistry) edited by E. Wunsch, vol. 15-I and II, THIEME, STUTTGART, 1974, or that of peptide synthesis in solid phase described by RD Merrifield in "Solid Phase Peptide Synthesis" (J. AM. CHEM. SOC.,<u style="single">45</u>, 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 DNAase I, then addition of EDTA and purification by extraction with the phenol / chloroform / isoamyl alcohol mixture ( 25/24/1) then with ether, - treatment 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, - 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, - the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the sought nucleic acid 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 β-cyanethyl phosphoramidite described in Bioorganic Chemistry 4; 274-325 (1986), - the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid by hybridization with an appropriate probe.

Another method for preparing the nucleotide sequences of the invention comprises the following steps: - The assembly of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the amino acid sequence of the natural polypeptide according to the principle described in Proc. Natl. Acad. Sci. USA, 80; 7461-7465, (1983), - the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid sought by hybridization with an appropriate probe.