EP3619327A1

EP3619327A1 - In vitro method for detecting and quantifying hiv-2 dna

Info

Publication number: EP3619327A1
Application number: EP18726743.0A
Authority: EP
Inventors: Adeline MELARD; Mélanie BERTINE; Véronique AVETTAND FENOEL; Florence DAMOND; Christine Rouzioux; Diane Descamps; Marie Gueudin; Jean-Christophe PLANTIER
Original assignee: Universite de Rouen; Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris 5 Rene Descartes; Universite Paris Diderot Paris 7; Universite Sorbonne Paris Nord Paris 13; Centre Hospitalier Universitaire de Rouen
Current assignee: Universite de Rouen; Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Sorbonne Paris Nord Paris 13; Centre Hospitalier Universitaire de Rouen; Universite Paris Cite
Priority date: 2017-05-05
Filing date: 2018-05-04
Publication date: 2020-03-11
Also published as: US11459623B2; FR3065967B1; US20200080160A1; WO2018202904A1; FR3065967A1

Abstract

The present invention relates to a method for detecting or quantifying human immunodeficiency virus 2 (HIV-2) deoxyribonucleic acid (DNA) in a sample containing DNA, which comprises: a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction of same comprising the DNA, with at least two primer and probe assemblies each comprising two primers and one marked probe, respectively, intended for detecting or quantifying HIV-2 DNA, in which at least one of the assemblies is chosen from the group made up of: an assembly comprising a primer comprising or consisting of a sequence of SEQ ID NO.: 1 or a sequence that is at least 90% identical with SEQ ID NO.: 1, a primer comprising or consisting of a sequence of SEQ ID NO.: 2 or a sequence that is 90% identical with SEQ ID NO.: 2 or the complements of these sequences, and a marked probe comprising or consisting of a sequence of SEQ ID NO.: 3, or a sequence that is at least 90% identical with SEQ ID NO.: 3 or the complement of these sequences, and an assembly comprising a primer comprising or consisting of a sequence of SEQ ID NO.: 4 or a sequence that is at least 90% identical with SEQ ID NO.: 4, a primer comprising or consisting of a sequence of SEQ ID NO.: 5 or a sequence that is at least 90% identical with SEQ ID NO.: 5 or the complements of these sequences, and a marked probe comprising or consisting of a sequence of SEQ ID NO.: 6, or a sequence that is at least 90% identical with SEQ ID NO.: 6 or the complement of these sequences; and b) determining from same the presence or absence and/or the amount of HIV-2 DNA in the biological sample.

Description

IN VITRO METHOD FOR DETECTING AND QUANTIFYING HIV-2 DNA

Field of the invention

The present invention relates to an in vitro method for the detection and quantification of human immunodeficiency virus 2 (VI H-2).

Technical background

Infection with HIV H - 2, mainly limited to West Africa, is characterized by a slow progression of the disease associated with a slow decrease in the number of CD4 ⁺ T lymphocytes, a low rate of transmission by pathway. sexually or vertically and has a lower viral replication than HIV-1.

In addition, individuals infected with HIV-2 have a natural resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), fusion inhibitors (eg, Enfuvirtide) and some protease inhibitors (PIs). . The treatment of individuals infected with HIV-2 therefore requires specific therapeutic management and different from individuals infected with VI H-1.

However, the detection of individuals infected with HIV-2 is difficult. Indeed, even in the absence of antiretroviral therapy, a high proportion of these individuals have an undetectable HIV-2 plasma RNA load. Thus, in the French national cohort of VI H-2 (1,086 patients in May 201 6), 71% of individuals who had never received antiretroviral therapy had viral loads of HIV-2 RNA less than 100 copies. / mL. In addition, data from infected individuals in West Africa revealed that in 46.5% of individuals who had never received antiretroviral therapy, HIV-2 RNA was undetectable (<1 0 copies / mL). ) and that in 35.8% of individuals, the HIV-2 RNA load was between 10 and 100 copies / mL.

In this context, HIV-2 DNA is currently considered to be the only detectable alternative marker in individuals whose H-2 VI RNA is undetectable.

This marker is particularly useful for confirming the diagnosis of mono or co-infection with HIV-1 in cases of high serological cross reactivity. In addition, the detection of HIV-2 DNA is essential for early diagnosis in children born to HIV-2 infected mothers. Finally, this marker may be useful for physiopathological studies of VI H-2 reservoirs. Several techniques for quantifying HIV-2 DNA are part of the state of the art, such as that described by Damond et al. (2001) J. Clin. Microbiol. 39: 4264-4268, in which the authors perform a real-time polymerase chain reaction (PCR) to amplify the gag region of the viral genome.

However, current techniques of HIV-2 DNA have difficulty quantifying HIV-2 DNA in group B. In addition, they lack sensitivity and are not always able to detect DNA. HIV-2 in some samples from infected individuals.

Moreover, no quantification test for HIV-2 DNA is currently available commercially.

Thus, the object of the invention is to provide a method for the detection and quantification of HIV-2 DNA to overcome these difficulties, in particular by improving the reproducibility, sensitivity and / or specificity of the methods of the invention. state of the art, particularly for the two endemic groups A and B of HIV-2.

Summary of the invention

The present invention results from the unexpected discovery, by the inventors, that a real-time PCR carried out with a set of primers of SEQ ID NO: 1 and 2 sequences and a probe of sequence SEQ ID NO: 3 , and a set of primers of SEQ ID NO: 4 and 5 and of a probe of sequence SEQ ID NO: 6, made it possible to reproducibly detect and quantify HIV-2 DNA of types A and B with a specificity of 100%, a detection limit of 3 copies / PCR and a limit of quantitation at 6 copies / PCR.

The present invention thus relates to a method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample comprising DNA comprising:

a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probes each comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of which is selected from the group consisting of: an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complementary of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement of these sequences, and

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or the complementary of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement of these sequences;

b) determining therefrom the presence or absence and / or amount of HIV-2 DNA in the biological sample.

In one embodiment of the invention, in the method for detecting or quantifying HIV-2 DNA as defined above, step a) of carrying out a polymerase chain reaction (PCR) in real time on the sample, or a fraction thereof comprising DNA, is carried out with

(i) at least 4 primers comprising or consisting of:

a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1 or the complement of these sequences, and

a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complement of these sequences, and

a sequence SEQ ID NO: 4 or a sequence having 90% identity with SEQ ID NO: 4 or the complement of these sequences, and

a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or the complement of these sequences, and

(ii) at least 2 labeled probes comprising or consisting respectively of:

a sequence SEQ ID NO: 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement thereof, and a sequence SEQ ID NO: 6, a sequence complementary to SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof.

In a particular embodiment of the method as defined above, at least one DNA virus or at least one DNA molecule having essentially no similarity in the sample or fraction thereof is added to the sample or fraction thereof. sequence with the genomic sequence of HIV-2, as well as the primers and probe necessary for its amplification and detection by real-time PCR, as an internal control of extraction and / or inhibition.

In another particular embodiment of the invention, the method for detecting or quantifying HIV-2 DNA as defined above further comprises determining or quantifying HIV-1 nucleic acids in a sample or fraction of it.

The present invention also relates to a kit or mixture for detecting or quantifying HIV-2 DNA, comprising:

a) at least two sets of primers and probe each respectively comprising two primers and one probe for the detection or quantification of HIV-2 DNA, preferably the primers and the probe being each present in a concentration; between 300 and 500 nM, in particular 400 nM, and at least one of which is selected from the group consisting of:

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complementary of these sequences, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 ( BHQ1) at its 3 'end, comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement of these sequences, and

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or complementary to these primers, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 (BHQ1) at its 3' end, comprising or consisting of a sequence SEQ ID NO : 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement of these sequences, and

b) at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2, as well as the primers and probes necessary for its amplification and detection by PCR in time actual, as an internal control of extraction and / or inhibition, and c) optionally additional reagents to perform a PCR.

In a particular embodiment of the invention, the kit or mixture as defined above comprises:

a) at least 4 primers, preferably at a concentration of between 300 and 500 nM, in particular 400 nM, comprising or consisting respectively of:

a sequence SEQ ID: NO 1 or a sequence having at least 90% identity with SEQ ID NO: 1 or the complement of these sequences, and

a2) at least 2 labeled probes, in particular with 6-carboxyfluorescein (FAM) at their 5 'end and Black Hole Quencher-1 (BHQ1) at their 3' end, preferably at a concentration of between 300 and 500 nM , in particular 400 nM, comprising or consisting respectively of:

a sequence SEQ ID NO 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NI: 3 or the complement thereof, and

a sequence SEQ ID NO: 6, a sequence complementary to SEQ ID NO:

6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof. In one embodiment of the invention, the kit or mixture as defined above further comprises primers and at least one labeled probe for detecting or quantifying HIV-1 nucleic acids.

The present invention also relates to the use of the kit or mixture as defined above, for detecting or quantifying the HIV-2 nucleic acids of a sample containing DNA.

The present invention also relates to a method, particularly an in vitro method, for diagnosing an HIV-2 infection, or determining an HIV-2 viral load, in an individual, comprising the steps of:

(a) performing the method for detecting or quantifying HIV-2 DNA in a biological sample taken from an individual as defined above;

(b) determine from it whether the individual is infected with HIV-2 or the HIV-2 viral load of the individual.

The present invention also relates to a method, particularly an in vitro method, for determining whether an individual is likely to benefit from treatment of an HIV-2 infection or a treatment adjustment, comprising performing the method for diagnose an HIV-2 infection, or determine a viral load of HIV-2, as defined above.

The present invention also relates to a method, particularly an in vitro method, for monitoring the HIV-2 viral load of an individual treated for HIV-2 infection, comprising performing the method for diagnosing HIV infection. -2, or determine a viral load of HIV-2, as defined above.

The present invention also provides nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs) and / or integrase inhibitors for use in the prevention or treatment of HIV-2 infection in a human subject. individual, wherein the individual has been determined to be susceptible to treatment of HIV-2 infection by the method as defined above. Detailed description of the invention

HIV-2 DNA

The human immunodeficiency virus 2 (HIV-2), the infection of which can lead to Acquired Immunodeficiency Syndrome (AIDS), is well known to those skilled in the art. HIV-2 is a member of the genus Lentivirus, which belongs to the family Retroviridae. Its replication cycle involves the entry of HIV-2 single-stranded RNA into a cell, reverse transcription to give single-stranded DNA, and then double-stranded DNA that is integrated into the genome of the host cell. The HIV-2 according to the invention may be of any group, in particular of group A, B or of non-endemic groups, in particular C, D, E or H. Advantageously, the method according to the invention detects or quantifies the Group A or B HIV-2 DNA in a sample with a specificity of the order of 100%.

By the term "HIV-2 DNA" is meant DNA that encodes the entire HIV-2 genome in whole or in part. The HIV-2 DNA to be detected or quantified according to the invention can thus be in particular a single-stranded DNA, especially a sense or antisense single-stranded DNA, or a double-stranded DNA.

Real-time PCR

Real-time polymerase chain reaction (PCR) is well known to those skilled in the art and is also known as quantitative PCR (qPCR).

Those skilled in the art can readily determine from a real-time PCR whether HIV-2 DNA is present, i.e., detect, and / or quantify HIV-2 DNA. Typically, in real time PCR a signal, usually a fluorescent signal, the intensity of which is a consequence of amplified DNA accumulation, is measured by the thermal cycler that performs the PCR. During the PCR, if the intensity of the measured signal, in particular the fluorescent signal, is greater than a signal threshold, generally the intensity of the background signal, it is deduced that the amplification has taken place, .e. that the HIV-2 DNA is present in the biological sample. Conversely, if no signal intensity greater than the background signal intensity is measured during the PCR, it is deduced either that no DNA molecule is present in the biological sample or that the amount of DNA is below the level of detection. In addition, the PCR cycle during which an intensity signal higher than the background signal intensity is measured, called the threshold cycle (CT). It is well known to those skilled in the art that the CT values are proportional to the logio of the initial amounts of DNA in the sample subjected to the real-time PCR. Therefore, the amount of DNA present, ie the DNA load of the biological sample, can easily be determined, if necessary by reference to a standard curve.

Many real-time PCR techniques, usually differentiated by the signal generation system and the labeled probes used, can be used according to the invention, such as Taqman or Molecular Beacons technologies (molecular beacons). Preferably, the PCR according to the invention is of Taqman type. Real-time Taqman PCR is well known to those skilled in the art and was originally described in 1965 by Holland et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7276-7280.

Briefly, the Taqman probes consist of a fluorophore covalently attached to the 5 'end of an oligonucleotide probe and another type of fluorophore called "quencher" located at the 3' end. The probe is such that the quencher turns off the fluorescence emitted by the fluorophore when it is excited by a thermocycler light source, this phenomenon being known as fluorescence resonance energy transfer or FRET (Fluorescence Resonance Energy Transfer). ). Thus, as long as the fluorophore and the quencher are nearby, i.e. are attached to the probe, the quenching inhibits any fluorescence signal. In addition, Taqman probes are designed so that they hybridize to a target within an amplified DNA region. In this way, when the thermostable polymerase used to perform the PCR extends the primer and synthesizes the nascent strand, its 5'-3 'exonuclease activity degrades the probe that hybridized to the target. The degradation of the probe causes the release of the fluorophore and breaks the proximity to the quencher, dissipating the quenching effect and thus triggering the fluorescence of the fluorophore. Thus, the fluorescence detected in the quantitative PCR thermocycler is directly proportional to the released fluorophore and the amount of DNA template present in the PCR.

Preferably, the real-time PCR according to the invention comprises the following thermocycling conditions:

2 minutes at 50 ° C, followed by

10 minutes at 95 ° C, followed by - 50 cycles of 95 ° C for 5 seconds and 60 ° C for 1 minute.

Advantageously, the thermocycling conditions defined above are compatible with or identical to those used for the detection and / or quantification of HIV-1 DNA, in particular using the GENERIC HIV DNA Cell test. Biocentric company (Grasse, France). Consequently, the method for detecting and quantifying HIV-2 DNA in a sample containing DNA according to the invention can be used in the same thermocycler, with the same computer program and even, if necessary, in the same amplification plate as samples intended for the detection or quantification of HIV - 1 DNA, or even in the same samples.

Primers and probes

As used herein, a "primer" is an oligonucleotide, preferably a DNA oligonucleotide, useful for initiating replication by a DNA polymerase, particularly a thermostable DNA polymerase. Preferably, the primers according to the invention do not comprise more than 50 nucleotides, more preferably not more than 40 nucleotides, and more preferably not more than 30 nucleotides.

As used herein, a "probe" is an oligonucleotide, preferably a DNA oligonucleotide, that can hybridize to amplified DNA molecules, i.e., amplimers, as part of PCR. in real time according to the invention. Preferably, the probes according to the invention do not comprise more than 50 nucleotides, more preferably no more than 40 nucleotides, and more preferably no more than 30 nucleotides. The labeled probe is such that a detectable signal, preferably fluorescence, is emitted and increases in intensity as a consequence of the accumulation of the amplified DNA molecules. More preferably, the probes according to the invention are labeled, in particular covalently labeled, with a first fluorophore and with a second fluorophore of type {(quencher)), the fluorophore-quencher pair being such that the quencher extinguishes the emission fluorescence emitted by the fluorophore. In this context, it is preferred that the fluorophore be attached at or near the 5 'end of the probe and that the quencher be attached at or near the 3' end of the probe. Many fluorophore-quencher pairs adapted according to the invention can be designed by those skilled in the art. As an example, the pairs fluorophore-quencher 6-carboxyfluorescein (FAM) -carboxytetramethylrhodamine (TAMRA) e 6 6-carboxyfluorescein (FAM) -Black Hole Quencher-1 (BHQ1) are suitable for labeled probes according to the invention. A particularly preferred fluorophore-quencher pair according to the invention is the 6-carboxyfluorescein (FAM) -Black Hole Quencher-1 (BHQ1) pair. Furthermore, it is also preferred that the two labeled probes according to the invention be labeled with the same fluorophore-quencher pair. Thus, it is particularly preferred according to the invention that the two labeled probes are labeled with a 6-carboxyfluorescein (FAM) at their 5 'ends and with a Black Hole Quencher-1 (BHQ1) at their 3' ends.

In the present invention, when a primer or probe according to the invention is said to "understand" a particular sequence, the primer or probe may also include additional sequences extending from the 5 'end and / or or the 3 'end of the particular sequence. In contrast, when a primer or probe according to the invention "consists of" or "consists of" a particular sequence, the primer or probe does not include additional sequences in addition to the particular sequence.

Preferably, the at least 4 primers according to the invention consist respectively of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, and the two probes labeled according to the invention consist of respectively in the sequences SEQ ID NO: 3 and SEQ ID NO: 6.

In the present invention, a "sequence having at least 90% identity with SEQ ID NO: X", in particular differs from SEQ ID NO: X by inserting, deleting or substituting at least one nucleotide. On the other hand, the percentage identity between two nucleotide sequences is defined here as the number of positions for which the bases are identical when the two sequences are optimally aligned, divided by the total number of bases of the longest of the two. sequences. Two sequences are said to be optimally aligned when the percentage of identity is maximum. Moreover, according to the usual practice of those skilled in the art, it is possible in certain cases to add a limited number of spaces in order to obtain an optimal alignment between the two sequences. Preferably, a sequence according to the invention having at least 90% identity with SEQ ID NO: 1, 2, 3, 4, 5 or 6 respectively has at least 95%, more preferably at least 98% identity with SEQ ID NO: 1, 2, 3, 4, 5 or 6.

The primers according to the invention comprising the sequences SEQ ID NO: 1 and SEQ ID NO: 2 or the sequences having at least 90% identity with SEQ ID NO: 1 and SEQ ID NO: 2 and the probe comprising or consisting of SEQ ID NO: 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement thereof, are used to detect a portion of the LTR region of the HIV-2 genome.

The primers according to the invention comprising the sequences SEQ ID NO: 4 and

SEQ ID NO: 5 or sequences having at least 90% identity with SEQ ID NO: 4 and SEQ ID NO: 5 and the probe comprising or consisting of the sequence SEQ ID NO: 6, a sequence complementary to the SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof, are useful for detecting a portion of the gag region of the HIV-2 genome.

As will be clear to those skilled in the art and by way of example, the sequence complementary to a sequence:

5'-AGCAGGTAGAGCCTGGGTGTT-3 '(SEQ ID NO: 1) is 5'-AACACCCAGGCTCTACCTGCT-3' (SEQ ID NO: 7);

- 5'-TCTTTAAGCAAGCAAGCGTGG-3 '(SEQ ID NO: 2) is 5'-CCACGCTTGCTTGCTTAAAGA-3' (SEQ ID NO: 8);

5'-CTTGGCCGGYRCTGGGCAGA-3 '(SEQ ID NO: 3) is 5'-TCTGCCCAGYRCCGGCCAAG-3' (SEQ ID NO: 9);

5'-GCGCGAGAAACTCCGTCTTG-3 '(SEQ ID NO: 4) is 5'-CAAGACGGAGTTTCTCGCGC-3' (SEQ ID NO: 10);

5'-TTCGCTGCCCACACAATATGTT-3 '(SEQ ID NO: 5) is 5'-AACATATTGTGTGGGCAGCGAA-3' (SEQ ID NO: 1 1);

5'-TAGGTTACGGCCCGGCGGAAAGA-3 '(SEQ ID NO: 6) is' CTTTCCGCCGGGCCGTAACCTA-3' (SEQ ID NO: 12);

Moreover, as will be clear to the skilled person the SEQ ID

NO: 3 (CTTGGCCGGYRCTGGGCAGA) is a so-called degenerate sequence wherein Y is C and T, and R is A and G.

Preferably, the primers and probes according to the invention are present in a concentration of between 300 and 500 nM each. Preferably also primers and probes are all present at the same concentration. Most preferably, the primers and probes are present at a concentration of 400 nM.

Advantageously, the use of primers and probes according to the invention in a real-time PCR (i) allows the specific detection and quantification of HIV-2 DNA relative to the nucleic acids of HIV-1. (ii) decreases the limit of detection and quantification of state - of - the - art tests, and (iii) allows better detection and quantification of Group B HIV - 2 DNA. Internal Control

In the present invention, the internal control of extraction and / or inhibition makes it possible in particular to validate the entire analytical process by real - time PCR, from extraction to amplification, without competition between HIV DNA. -2 and internal control, including for weak values.

By the term "internal extraction and / or inhibition control" is meant here an exogenous DNA or nucleic acid molecule, having essentially no sequence similarity with the human genome or with pathogens affecting the humans. Preferably, the internal extraction and / or inhibition control according to the invention is a DNA molecule.

Preferably, the internal control according to the invention as well as the primers and probe necessary for its amplification and detection by real-time PCR are added before the extraction.

Preferably, the internal extraction and / or inhibition control according to the invention is labeled with a fluorochrome. More preferably, the internal control of extraction and / or inhibition according to the invention is marked by the fluorochrome Yellow Dye ref. DICD-YD-L1 00.

Sample containing DNA

In the present invention a "sample containing DNA" relates to any sample, in particular solid, fluid or liquid, likely to contain HIV-2 DNA. The sample containing the DNA according to the invention may in particular be a biological sample or a sample, in particular of experimental type, containing cells or fractions of cells infected in vitro by HIV-2. Preferably, the biological sample according to the invention is derived from a sample taken from an individual, in particular a human individual, and may especially be a whole blood sample, a plasma sample, a serum sample, a seminal sample. , a sample of peripheral blood mononuclear cells (PBMC), or fractions thereof, or a sample of leukocytes, or fractions thereof.

As will be clear to those skilled in the art, the sample containing the DNA according to the invention may have been treated, in particular after being taken from the individual, before being subjected to a real-time PCR according to US Pat. 'invention. Such treatments include centrifugation, treatment with an anticoagulant such as EDTA, or concentration, purification or extraction of DNA. Thus, for example, a biological sample according to the invention may be a DNA solution of a sample, such as a whole blood sample, taken from a human individual.

Kit and mix

By the term "kit" it is understood that one or more of the kit components may be packaged or compartmentalized separately from the rest of the components.

By the term "mixture" it is understood that all components of the mixture are in a single compartment. Preferably, the mixture according to the invention is a PCR mixture.

Additional reagents with respect to the primers and probes according to the invention can be easily designed by those skilled in the art and can include, in particular, a thermostable DNA polymerase, a reverse transcriptase, dNTPs, salts, in particular Mn ²⁺ and Mg ²⁺ , and buffers.

Treatment of HIV-2 infection

In the present invention, "treatment of HIV-2 infection" refers to any treatment that prevents or treats an HIV-2 infection, including reducing the HIV-2 viral load or rendering it undetectable. .

Preferably, the treatment of an HIV-2 infection according to the invention is selected from the group consisting of antiretroviral therapy (ART), in particular using a nucleoside reverse transcriptase inhibitor (NRTI). , a protease inhibitor (PI) and / or an integrase inhibitor, an immunomodulator, a molecule capable of acting at the level of the immune control points, a monoclonal antibody, a molecule anti -Late with an epigenetic mechanism of action and a vaccine.

The present invention will be further explained, without limitation, with the aid of the following figures and example.

Description of figures

Figure 1

FIG. 1 represents the standard curve of the threshold cycle (Cr) (vertical axis) determined by the implementation of a real-time PCR according to the invention as a function of the number of HIV-2 DNA copies introduced (axis horizontal, logarithmic scale). Median values and 25% and 75% interquartile ranges of Cr are indicated.

Figures 2 and 3

FIGS. 2A-2B show the logio of the copy number of HIV-2 DNA obtained by automatic extraction from 10 ⁶ PBMC (FIG. 2A) or leukocytes (FIG. 2B) (vertical axis) as a function of log 10 of the number copies of HIV-2 DNA obtained by manual extraction from 10 ⁶ PBMC (Figure 2A) or leukocytes (Figure 2B) (horizontal axis), the PBMCs being derived from cell pellets (Figure 2A) and leukocytes from whole blood (Figure 2B). The correlation coefficient (r) and significance threshold (p) are represented.

Figures 4 and 5

Figures 4 and 5 represent the amount of total HIV-2 DNA (vertical axis, log 10 copy number for 10 ⁶ PBMC) as a function of the amount of HIV-2 RNA (horizontal axis, in log 10 of the number of copies per mL (Figure 3) or in copy-number strata per mL (Figure 4)) for HIV-2 group A-treated patients (black triangles), group A non-HIV-2 positive patients treated (white triangles), group B HIV-2 positive patients treated (black circles) and group B HIV-2 positive patients (white circles). An arbitrary value of 1, 60 logio copies / mL was attributed to samples with undetectable plasma viral load.

EXAMPLE

Materials and methods

Biological samples according to the invention

Aliquots of frozen peripheral blood mononuclear cells (PBMCs) from 63 HIV-2 infected patients included in the French national cohort of HIV-2 (ANRS C05) were selected based on viral genotype and HIV RNA load Plasma -2 determined as described in Avettand-Fenoel et al. (2014) J. Clin. Microbiol. 52: 301-7-3022. Written consent was obtained from all patients at the time of inclusion in the cohort. Of these 63 patients, 71% (n = 45) were treated with antiretroviral therapy, and for the viral group, determined as described in Charpentier et al. (2014) AIDS Lond Engl 28: 1 1 61 -1 1 69, 35 of them had group A virus (71% treated) and 28 had group B virus (71% treated). The selected samples had the following characteristics: HIV-2 RNA load <40 copies / ml (n = 28; 15 from group A and 1 from group B), 40 to 500 copies / ml (n = 18; Group A and Group B), 500-5000 copies / ml (n = 9; 4 of group A and 5 of group B) and 5000-50000 copies / ml (n = 8, 6 of group A and 2). group B). These samples were used to evaluate the clinical performance of the method according to the invention. Eleven whole blood samples from patients infected with HIV-2 (6 group A and 5 group B) were also used to evaluate the process performance on whole blood.

Blood samples from 30 HIV-negative individuals, 40 M-group HIV-1 positive patients and 10 Group O HIV-1 positive patients were tested to evaluate the specificity of the method according to the invention.

Detection and quantification of HIV-2 DNA according to the invention

Several extraction methods were used for whole blood and cell pellets, according to the laboratory among the three (A, B and C) involved in this study. For whole blood, the total DNA was extracted from 200 μΙ using the NucleoSpin® Blood Kit (Macherey-Nagel, Dûren, Germany) in laboratories A and B and the QIAsymphony DSP DNA mini kit (Qiagen, Courtaboeuf , France) in laboratory C. For cell pellets, the QIAamp DNA mini kit and the QIAsymphony DSP DNA mini kit were used for extraction of total DNA from 3 to 5 million cells in laboratories A and C respectively.

In order to normalize the quantification of H - 2 VI DNA, the amount of total DNA in extracts was determined spectrophotometrically (Nanodrop, Thermo Scientific, Wilmington, NC, USA) (Labs A and B) or by quantitation. of the albumin gene (laboratory C) using the LightCycler ® FastStart DNA Master Hydroprobe kit (Roche, Mannheim, Germany) and serial dilutions of human genomic DNA (Roche) as a standard (Laurendeau et al. (1,999) Clin Chem 45: 982-986, Dehee (2001) J. Med Virol 65: 543-552).

The quantification method according to the invention is based in particular on a Taqman type triplex PCR approach targeting the consensus regions conserved in the long repetitive terminal sequence (LTR) and in the gag gene. It includes an internal control (Ye // ow Dye universal DNA extraction and inhibition control, Diagenode, Liège, Belgium) added before extraction.

The sense and antisense primers for the LTR region are 5'-

AGCAGGTAGAGCCTGGGTGTT-3 '(SEQ ID NO: 1) and 5'-TCTTTAAGC AAGC AAGCGTGG-3' (SEQ ID NO: 2) (Rouet et al J. Clin Microbiol 42: 41 47-53), with a probe internal 5 '-FAM-CTTGGCCGGYRCTGGGCAGA-BH Q 1 -3', (SEQ ID NO: 3), where FAM stands for carboxyfluorescein and BHQ 1 stands for black hole quencher I.

The sense and antisense primers for the gag region are 5'-GCGCGAGAAACTCCGTCTTG-3 '(SEQ ID NO: 4) and 5'-TTCGCTGCCC AC AC AATATGTT-3', respectively (SEQ ID NO: 5), with an internal probe. 5'-FAM-TAGGTTACGGCCCGGCGGAAAGA-BHQ 1 -3 '(SEQ ID NO: 6) (Damond et al (2005) J. Clin Microbiol 43: 4234-6).

Table 1: Primers and probes used for PCR analysis

LTR Gag

Primers 5'-AGCAGGTAGAGCCTGGGTGTT-3 '5'-GCGCGAGAAACTCCGTCTTG-3' (SEQ sense (SEQ ID NO: 1) ID NO: 4)

5'-TCTTT AAGC AAGC AAGCGTGG-3 '5'-TTCGCTGCCCACACAATATGTT-3' primers (SEQ antisense (SEQ ID NO: 2) ID NO: 5)

Probe 5'-CTTGGCCGGYRCTGGGCAGA-3 '5'-TAGGTTACGGCCCGGCGGAAAGA-3'

(SEQ ID NO: 3) (SEQ ID NO: 6) The reaction mixture consists of a volume of 50 μl containing the DNA extract (20 μl), primers and probes for HIV-2 (400 nM each), the primers and the probe for internal control (1). μΙ) and PCR buffer IX (2X qPCR MasterMixPlus, Eurogentec, Seraing, Belgium).

The thermocycling conditions are as follows: 2 minutes at 50 ° C. and 10 minutes at 95 ° C., followed by 50 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The amplification and data acquisition were carried out using the CFX96 system (Biorad, Hercules, CA, USA) (Laboratory A) and the real-time PCR system TaqMan ABI 7900 and 7500 (Applied Biosystems, Courtaboeuf, France). France) (Laboratories B and C respectively). The logio of the number of targets initially present is proportional to the threshold cycle (Cr) and has been determined from the external standard curve.

DNA from HIV-2 infected cells (NIH-Z strain) (Advanced Biotechnologies Inc, Eldersburg, MD, United States of America) is used as an external standard. This standard, evaluated at 131,300 copies / μl using a previously described test (Damond et al (2005) J Clin Microbiol 43: 4234-4236), was first diluted in 200 ng. / μΙ of human genomic DNA (Promega, Madison, Winsconsin, United States of America) at a theoretical concentration of 60 000 ∞ρΐθ5 / 20μΙ, followed by a series of dilutions at 1/1 0 in 25 ng / μΙ d Human genomic DNA at concentrations ranging from 6,000 copies / 20 μl to a final dilution of 2 copies / 20 μl.

HIV-2 DNA levels were first reported as the HIV-2 DNA copy number / PCR. The number of copies of HIV-2 DNA ug total DNA was then calculated using the concentration of extract and the final results were reported as the number of copies / 1 0 ⁶ cells. The formula used to convert these results is: HIV-2 DNA (1 μg copies of total DNA) × 1,000,000 / 150,000 = HIV-2 DNA (copies / 10 ⁶ cells) (Avettand-Fénoël et al. (2009) J. Med Virol 81: 217-223, Dib et al (1996) Nature 380: 521-54). Determination of the analytical performance of the method for detecting and quantifying HIV-2 DNA according to the invention

Specificity was determined by testing of samples of HIV-negative subjects (10 per laboratory), M-group HIV-1 positive (laboratory A) and group O HIV-1 positive (laboratory). B). Linearity was evaluated in the three laboratories using serial dilutions of the external standard at 6000, 600, 60, and 6 copies / 20Ml (22 PCR).

Analytical sensitivity was determined by testing dilutions of the external standard at 10, 6, 4, 3, and 2 copies / 20Ml (replicated 20-fold) (laboratory A and laboratory B).

Intra-PCR reproducibility was determined by testing the external standard at 6000, 600, 60, and 6 copies / 20Ml dilutions (replicated 10 times for each dilution) (laboratory B).

To determine inter-PCR reproducibility, an HIV-2-positive control was prepared by diluting a DNA extract from cell cultures of a group A HIV-2 isolate (GenBank Accelerator Number M). 5390, SEQ ID NO: 1 3) and quantified at 2.13 log10 copies / 20 μM by a previously described assay (Damond et al (2005) J. Clin Microbiol 43: 4234-4236). This solution was analyzed in the three laboratories in separate PCRs (n = 24).

Manual and automated extractions were compared using cell pellets and whole blood; 15 PBMC pellets (8 from group A and 7 from group B) and 11 whole blood samples (6 from group A and 5 from group B) were extracted and quantified in parallel in laboratories A (manual) and C (automated). Statistical analysis

Comparisons between the groups were made with the Mann-Whitney test. Pearson correlation coefficients were calculated to estimate the relationship between O values and the logio of HIV-2 DNA copy number / PCR, the relationship between HIV-2 and HIV DNA loadings. HIV-2 RNA, and to compare manual extractions with automated extractions. Anova test was performed to evaluate the quantification values of HIV-2 DNA according to the stratification of the HIV-2 RNA quantification values.

Results

Analytical performances of the detection and quantification method according to the invention

The 50 positive or HIV-1 DNA samples and 30 HIV-negative DNA samples are negative in the assay, giving a specificity of 100%. The standard curve shows a strong linearity between the CT values and the logio of the HIV-2 DNA copy number / PCR, with a limit of 6 copies / PCR quantification (Figure 1). The median correlation coefficient is 0.997 (in a range 0.982 to 1), and the median slope is -3.45 (in a range of -3.1 to -3.64).

The analytical sensitivity of the assay is 100% at 4 copies / PCR, 95% at 3 copies / PCR and 85% at 2 copies / PCR.

Intra-PCR reproducibility was evaluated using the external standard with theoretical concentrations of 6000, 600, 60 and 6 copies / PCR (log 10 copies / PCR respectively 3.78, 2.78, 1, 78 and 0.78). The inventors obtained an average of 3.80 log10 copies / PCR for the expected value of 3.78 log10 copies / PCR with an intra-PCR coefficient of variation (CV) of 1.03% as well as average values of 2.79, 1, 83 and 0.85 log10 copies / PCR respectively for expected concentrations of 2.78, 1, 78, and 0.78 log10 copies / PCR, and intra- PCR resumes of 1.60 % 3.43% and 27.02%, respectively.

The positive control was evaluated at 2.19 log10 copies / PCR for inter-PCR assays performed in the three laboratories, with a CV of 5.10%.

Manual extractions and automated extractions were compared using samples extracted and quantified in parallel in laboratories A and C, respectively. Thus, the median DNA of HIV-2 obtained from May 1 cell pellets were 2.34 logio copies / 10 ⁶ PBMC with manual extraction and 2.29 in logio copies / 10 ⁶ PBMC with automated extraction, with a median of 0.22 log 10 differences and a correlation coefficient of 0.97 (95% CI = [0.92, 0.99], ρΟ, ΟΟΟΙ). Median values obtained from whole blood samples were 2.05 log10 copies / 10 ⁶ leucocytes with manual extraction and 1.73 log10 copies / 10 ⁶ leukocytes with automated extraction, with median differences 0.3 log 10 and a correlation coefficient of 0.96 (95% CI = [0.85, 0.99], p <0.0001) (Figures 2 and 3). Clinical performance

Clinical performance was assessed in laboratory C. All samples from patients infected with HIV-2 were validated according to the instructions of the manufacturer of the internal control. 122 to 1000 ng (median 548 ng) of total DNA per PCR well was analyzed, depending on the total DNA concentration in the extracts. VI H-2 DNA was detectable in all 63 patients. HIV-2 DNA was detectable but not quantifiable (<6 copies / PCR) for 20 patients (32%) and quantifiable (> 6 copies / PCR) for 43 patients (68%) with a DNA load of HIV-2 median of 2.45 in log 10 copies / 10 ⁶ PBMC (interquartile range [PIQ] = 2.15-3.00 log 10). For the 20 patients with detectable but not quantifiable HIV-2 DNA, the same DNA extracts were again analyzed using 2-6 replication PCRs. Eighteen samples gave positive results in all replicates, one sample had two out of three positive results and one had three out of four positive results, at a level below 6 copies per PCR.

Of the 35 group A samples, HIV-2 DNA was quantifiable in 21 samples (60%), with a median load of 2.56 log 10 copies / 10 ⁶ PBMCs (PIQ = 2.29-3, 03 logio). Of the 28 group B samples, HIV-2 DNA was quantifiable in 20 samples (71%), with a median load of 2.27 log 10 copies / 10 ⁶ PBMCs (PIQ = 1.97-2.81). logio). There was no difference between groups A and B with respect to the proportion of patients with HIV-2 DNA load below the limit of quantification (p = 0.79) or in the load median (2.56 vs 2.27 log10 copies / 10 ⁶ PBMC respectively, p = 0.17) when this load was quantifiable.

Of the 18 patients who had never received antiretroviral therapy, HIV-2 DNA was quantifiable in 10 of them (56%), with a median load of 2.08 log 10 copies / 10 ⁶ PBMCs (PIQs). = 1.88-2.28 log). Of the 45 patients who were treated with antiretrovirals, HIV-2 DNA was quantifiable in 33 of them (73%), with a median load of 2.60 log 10 copies / 10 ⁶ PBMCs (PIQ = 2.26-3.09 log 10). There was no difference in the proportion of patients with HIV-2 DNA load below the limit of quantification between patients who had never received antiretroviral therapy and treated patients (p = 0.23 for all patients, p = 0.74 for group A, and 0.65 for group B). When quantifiable, the median HIV-2 DNA load was significantly higher in antiretroviral-treated patients compared to those who had never been treated (p = 0.003 for all patients, p = 0.068 for group A, and 0.03 for group B). When it was quantifiable, the HIV-2 DNA load correlated with the HIV-2 RNA load (r = 0.68, 95% CI [0.4-0.8], ρΟ, ΟΟΟΙ for the whole group and r = 0.73, 95% CI [0.4-0.9], p <0.0002 for the treated patients) (Figure 4). The proportions of patients with HIV-2 DNA load below the limit of quantification were 46%, 39%, 0%, and 0%, respectively, for HIV-2 RNA load <40 copies / ml, from 40 to 500 copies / ml, from 500 to 5000 copies / ml, and from 5,000 to 50,000 copies / ml. When they were quantifiable, the median HIV-2 DNA (PIQ) loads were 2.26 in log10 (2.02-2.56 log 10), 2.37 in log 10 (2.01 -2, 50 log 10), 2.74 log 10 (2.7-3.24 log 10) and 3.42 log 10 copies / 10 ⁶ PBMC (3.06-3.58 log 10) respectively (FIG. 5). HIV-2 DNA loads were significantly different depending on the stratification of the HIV-2 RNA loads (Anova Test, p <0.0001). A comparison between subgroups (group A / group B, antiretroviral / untreated patients) versus HIV-2 RNA load was not relevant because of low numbers of patients in each subgroup.

Conclusion

HIV-2 infection is very different from that of HIV-1, particularly with respect to its slower progression, its therapeutic management and the level of genetic diversity of HIV-2. Specific molecular methods are therefore needed for the diagnosis and monitoring of HIV-2 infection (either alone or in the context of HIV-1 co-infection), for the diagnosis of infants born to mothers positive for HIV-2, as well as for the study of the HIV-2 reservoir. Plasma viraemia is undetectable in a large number of patients infected with HIV-2 in the absence of antiretroviral therapy, particularly in those with high CD4 ⁺ T cell counts. HIV-2 DNA may be the only detectable marker in these patients.

The object of the present invention is therefore in particular to develop a quantitative assay of HIV-2 DNA highly sensitive and easy to implement in developing countries where HIV-2 is endemic.

The method of detection and quantification according to the invention has a specificity of 100% which means that the primers of HIV-2 have not hybridized with the genome of HIV-1. This method also has good linearity (6 to 6000 copies / PCR) as well as good intra-PCR reproducibility. The inventors have elsewhere shown good inter-laboratory reproducibility. Moreover, the detection and quantification method according to the invention which may include an external standard for quantification makes it possible to improve reliability and comparisons between different studies. The excellent sensitivity (95% detection limit: 3 copies / PCR) is both useful for clinical diagnosis and for physiopathological studies.

Both manual and automatic extraction methods, as well as two real - time PCR instruments, have been validated for compatibility with local practices in resource - constrained countries. Finally, the inventors have also shown that the process according to the invention can be carried out both on blood cell pellets and on whole blood samples.

The inventors used the same protocol as the Generic HIV DNA cell kit (Biocentric, Bandol, France) used to detect and quantify HIV-1 DNA. This test is currently being used successfully in several resource-limited countries. This thus makes it easier to use the test according to the invention for either HIV-2 alone or together with HIV-1, in the same PCR. This reduces analytical costs by increasing the number of PCR samples and facilitates the molecular diagnosis of HIV-1 and HIV-2 dual infection in the same sample or the diagnosis of HIV infection in patients with HIV. infants.

HIV-2 DNA could be detected in the blood cells of all patients (infected with either HIV-2 group A or group B HIV-2); the sensitivity of the method according to the invention was thus improved over previous assays which reported that HIV-2 DNA was undetectable in 17% (5/29) of HIV-2 infected patient specimens ( Damond et al (2001) J. Clin Microbiol 39: 4264-4268).

The addition of an internal control, which was absent in previous tests

(Damond et al (2001) J. Clin Microbiol 39: 4264-4268, Gueudin et al (2005) Meth Mol Biol 304: 215-220) allows the validation of the analytical process.

The inventors have also shown that patients infected with HIV-2 receiving antiretroviral therapy had higher HIV-2 DNA loads than patients who had never received antiretroviral therapy, probably because the patients treated had a more advanced disease that required the initiation of antiretroviral therapy. Among the treated patients, a higher HIV-2 DNA viral load is correlated with higher HIV-2 RNA loads, similar to what was reported for infections with HIV-2. HIV-1 (Visseaux et al (201 6) 23th Conference on Retroviruses and Opportunism Infections Abstract 2 14. Boston, Massachusetts, USA).

Quantification of HIV-2 DNA levels could also provide information about the HIV-2 reservoir, just as the HIV-1 DNA load has been described as a relevant marker of the HIV-1 reservoir . In fact, the DNA load of HIV-1 is predictive of immunological and clinical progression, regardless of the amount of CD4 ⁺ lymphocytes and the viral RNA load. HIV-1 DNA levels vary according to the stage of HIV infection, with the highest levels found during primary HIV infection and during AIDS. In addition, quantification of HIV-1 DNA has allowed for reservoir studies in different types of cohorts of patients: long-term non-progressors, elite controllers, untreated chronic infected patients, and controllers. after treatment. This paves the way for a better understanding of the natural history of HIV - 1 infection.

In conclusion, the inventors have developed a method for the detection and quantification (or assay) of HIV-2 DNA which has good analytical performance as well as good clinical sensitivity. This method is particularly effective for the more frequent HIV-2 groups A and B compared to the previously described tests (Damond et al (2001) J. Clin Microbiol 39: 4264-4268, Gueudin et al. (2008) AIDS Lond, Engl 22: 21-215). The method according to the invention has been validated over a wide and well characterized range of patient samples. This method is easy to implement and is suitable for use in resource-limited countries in which multiple variants of HIV-2 circulate. It can also be particularly useful for the diagnosis of HIV-2 in infants born to HIV-infected mothers, for the diagnosis of mono-infections or co-infections with HIV-1, which is important because of the therapeutic consequences and monitoring. Finally, this method is also useful in HIV-2 reservoir pathogenesis studies, exploring new insights into the natural history of HIV-2 infection at different stages. , and improved opportunities for clinical studies in treated patients.

Claims

A method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample containing DNA comprising:

a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probes each comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of which is selected from the group consisting of:

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complementary of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity with the SEQ ID NO: 3 or the complement of these sequences, and an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or the complementary of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence with at least 90% identity with SEQ ID NO: 6 or the complement of these sequences, and b) determine therefrom the presence or absence and / or amount of HIV-2 DNA in the biological sample. The method according to claim 1, wherein the step a) of performing a real time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, is carried out with

(i) at least 4 primers comprising or consisting of:

(ii) at least 2 labeled probes comprising or consisting respectively of:

a sequence SEQ ID NO: 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement thereof, and

a sequence SEQ ID NO: 6, a sequence complementary to SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof.

The method of claim 1 or 2, wherein the sample containing DNA is a biological sample or a sample comprising cells infected in vitro by HIV-2.

The method according to any of claims 1 to 3, wherein the primers and probes are present in a concentration of between 300 and 500 nM each.

The method according to any one of claims 1 to 4, wherein the primers are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the probes labeled consist of respectively in the sequences SEQ ID NO: 3 and SEQ ID NO: 6.

The method according to any one of claims 1 to 5, wherein the labeled probes are labeled with 6-carboxyfluorescein (FAM) at their 5 'end and with Black Hole Quencher-1 (BHQ 1) at their 3 end. '.

The method according to any one of claims 1 to 6, wherein at least one DNA virus or at least one DNA molecule having essentially no similarity is added to the sample or fraction thereof. sequence with the genomic sequence of HIV-2, as well as primers and probes necessary for its amplification and detection by real-time PCR, as an internal extraction and / or inhibition control.

The method of any one of claims 1 to 7, wherein the PCR comprises the following thermocycling conditions:

- 2 minutes at 50 ° C, followed by

- 1 0 minutes at 95 ° C, followed by

50 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute.

The method of any one of claims 1 to 8, further comprising determining or quantifying HIV-1 nucleic acids in the sample or a fraction thereof.

A kit or mixture for detecting or quantifying HIV - 2 DNA comprising: a) at least two sets of primers and probes each of which comprises two primers and one probe respectively for detecting or quantifying the HIV-2 DNA, preferably the primers and the probe being each present in a concentration of between 300 and 500 πΜ, e † at least one of which is selected from the group consisting of:

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complementary of these sequences, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 ( BHQ 1) at its 3 'end, comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement of these sequences, and

an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or the complementary of these primers, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 ( BHQ 1) at its 3 'end, comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement of these sequences, and

b) at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2, as well as the primers and probes necessary for its amplification and detection by PCR in time as an internal control of extraction and / or inhibition, and

c) optionally additional reagents to perform a PCR. Kit or mixture according to claim 10, comprising:

al) at least 4 primers, preferably at a concentration between 300 and 500 nM, comprising or consisting respectively of: a sequence SEQ ID: NO 1 or a sequence having at least 90% identity with SEQ ID NO: 1 or the complement of these sequences, and

a2) at least 2 labeled probes, in particular with 6-carboxyfluorescein (FAM) at their 5 'end and Black Hole Quencher-1 (BHQ 1) at their 3' end, preferably at a concentration between 300 and 500 nM, comprising or consisting respectively of:

a sequence SEQ ID NO 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NI: 3 or the complement thereof, and a sequence SEQ ID NO : 6, a sequence complementary to SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof.

The kit or mixture of claim 10 or 11, further comprising primers and at least one labeled probe for detecting or quantifying HIV-1 nucleic acids.

Use of the kit or mixture as defined in any one of claims 10 to 12 for detecting or quantifying HIV-2 nucleic acids of a sample containing DNA.

An in vitro method for diagnosing an HIV-2 infection, or determining an HIV-2 viral load, in an individual, comprising the steps of: (a) performing the method for detecting or quantifying HIV-2 DNA in a biological sample taken from an individual as defined in claims 1 to 9;

15. In vitro method for monitoring the HIV-2 viral load of an individual treated for HIV-2 infection, comprising performing the method for diagnosing HIV-2 infection, or determining a viral load of HIV -2, according to claim 14.

16. In vitro method for determining whether an individual is likely to benefit from treatment of HIV-2 infection or treatment adjustment, including performing the method for diagnosing HIV-2 infection, or determining a viral load of HIV-2, according to the claim

1 4.

17.Nucleoside reverse transcriptase inhibitors (INTs), protease inhibitors (PIs) and / or integrase inhibitors for use in the prevention or treatment of HIV-2 infection in an individual, in which the individual has been determined to be amenable to treatment of an HIV-2 infection according to claim 1 6.